Imidazole derivatives differentially destabilize the low pH conformation of lysozyme through weak electrostatic interactions

Abstract

The structure–stability–function paradigm of proteins is highly governed by the pre-existence of repulsive and attractive non-covalent interactions. Electrostatic interactions with charged solvent molecules at the protein surface can contribute significantly to their stability. In order to unravel the electrostatic contributions of the positively charged species on a protein, we have studied the interactions of the imidazole derivatives (imidazole, histidine and histamine) with Hen Egg Lysozyme (HEL) in the pH range 4–2. At acidic pH, these compounds are reported to form charge transfer complexes (CTCs) with HEL through their protonated imidazole moiety with indole ring of protein's tryptophan. Although literature is available on the molecular geometry and functional roles of CTCs, no knowledge is available on the electrostatic interactions and stability attenuation features of these complexes. To unravel the stability aspects of HEL upon binding to imidazole derivatives, we have performed pH dependent stability and structural studies using various biophysical techniques. Our results evidenced a stability attenuation of HEL in the pH range 4–2, both in its apo conformations and also in complex with imidazole derivatives. Moreover, at pH values 4 and 3 all these positive charged compounds destabilized HEL by a T_m of 5–7 °C, leaving the pH 2 conformation untouched. Structural analysis suggested that interactive networks of negatively charged Glu/Asp residues of the protein with CTC forming compounds is responsible for such differential stability attenuation. We believe that our results are handy to the researchers in deciphering the contributions of weak non-covalent interactions in biomolecular recognition processes.

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Introduction

Elucidating the role of intrinsic structural elements and the environmental factors that govern protein stability is a highly challenging and fascinating contemporary research area of molecular biophysics. Proteins reside in their native structure with a global free energy minimum due to pre-existence of repulsive/attractive electrostatic and hydrophobic interactions.^1–4 Apart from the covalent polypeptide linkage, the contributions of the non-covalent interactions are crucial in providing the overall stability and structural integrity to proteins. Indeed, these non-covalent interactions play pivotal roles during biomolecular recognition processes.^5–7

Electrostatic interactions are a class of non-covalent interaction, experienced between the pair of atoms containing a net charge. The net charge carried by the solvent ions is crucial to the protein stability as they interact with the exposed side chains of the protein's charged residues, which in turn is regulated by the pK_a value of that particular amino acid. Similarly, formation of the charge transfer complexes (CTCs) between the protonated imidazole (histidine – His) and indole ring (tryptophan – Trp) at acidic pH in proteins also play regulatory structural/functional roles.^8,9 Formation of CTCs can be monitored by specific absorption peaks between the 240–320 nm in UV absorption spectrum and a decrease in the fluorescence intensity of the Trp residue emission.^10–13 Furthermore, the high density positive charge of these imidazole derivatives at low pH values is a potential source of electrostatic interactions with the ionizable side chains of negatively charged residues in the proteins.

Hen egg lysozyme (HEL) is a muramidase, or glycoside hydrolase protein, present in most of the bird eggs, secretion like tears, saliva and as well as in other body fluids.¹⁴ HEL is one of the well characterized proteins to unravel the structure–stability–folding–function paradigm.^15–19 HEL has a very stable structural fold, comprising of both helical and sheet secondary structures, although the helical content is predominant (Fig. 1A). It contains six tryptophan residues at positions 28, 62, 63, 108, 111 and 123, with W62, W63 and W108 are located at the active site of protein.^17,20,21 HEL had been chosen for several decades as a model protein to understand the charge transfer complex formation owing to its (i) commendable structural stability at acidic pH values and (ii) presence of solvent exposed tryptophans that can act as ideal electron donors. Various studies had been reported on the formation of charge transfer complexes between HEL and the imidazole like compounds.^21,22 Literature also depicted that such a CTC formation in HEL with several of the imidazole derivatives had significantly attenuated its catalytic function.^10,23 Apart from the Trp residues (W62, W63) at catalytic site of HEL (site-I, Fig. 1B), a solvent exposed Tyr residue (Y23 (ref. 24–26)) that is lying on the top of the buried indole rings of W28 and W111 was also reported to form a secondary site of CTC interactions in HEL (site-II, Fig. 1B). However, till date no detailed study has been reported regarding the electrostatic contributions of these charged derivatives on HEL stability.

Fig. 1 (A) Cartoon representation of the structural topology of HEL (PDB ID: 193L) produced using PyMol graphics software; the pink spheres represent the positions of the tryptophan residues. (B) Representation of the side chain networks and the presumed site-I and site-II of the charge transfer complex formation on the structure of HEL (PDB ID: 193L). The distances between the nearby aromatic rings/other interacting side chain (<5.5 Å) with the CTC forming residues W62 and Y23 are marked. The W63–W108 inter-tryptophanyl energy transfer (<7.8 Å), that is crucial in fluorescence intensity attenuation upon CTC formation is also highlighted. The figures were generated using PyMol molecular graphics software. (C) Protonated form of the imidazole derivatives (imidazole, histidine and histamine); the structures were drawn using ChemSketch.

In order to throw light on the stability modulations of the proteins upon association of weak electrostatic interactions with the imidazolium ions, we have studied the energetic characteristics of HEL complexes with imidazole derivatives (imidazole, histidine and histamine) (Fig. 1C) at acidic pH (in the pH range 4 to 2) using a variety of spectroscopic and calorimetric techniques such as Differential Scanning Calorimetry (DSC), Circular Dichroism (CD), fluorescence spectroscopy and NMR experiments. Our comprehensive biophysical analysis suggested that, weak electrostatic interactions indeed attenuate the stability characteristics of HEL in a pH dependent manner and is dependent on the side chain pK_a values of the ionizable groups.

Materials & methods

Sample preparation

Ultra-pure grade hen egg lysozyme (0663-10G) was purchased from Amresco Company USA; imidazole, histidine, histamine and other lab chemicals from Sisco Research Laboratory (SRL Chem) India. Lysozyme powder was dissolved to a concentration of 10 mg mL⁻¹ in 20 mM glycine–HCl buffer (pH 2, 3, and 4) and protein samples were dialyzed in the different pH buffers for 6 hours using Spectra dialysis membrane (Cutoff 2000 Da) and filtered with 0.22 micron syringe filter. The concentration of the samples was determined spectrophotometrically using ε₂₈₀ = 36 000 M⁻¹ cm⁻¹ and the protein samples were stored at −80 °C. The purity of the protein was assessed from the single band obtained on SDS-PAGE. Imidazole, histidine, histamine and NaCl solutions were prepared by dissolving the powder in 100 mM Glycine–HCl buffer at respective pH (2.0, 3.0 and 4.0). All the fluorescence, CD and DSC experiments were performed using a ligand concentration of 100 mM with the same parameters as that of apo.

Circular-dichroism (CD) spectroscopy

Far-UV CD experiments were performed on Chirascan applied photophysics CD spectrophotometer at 25 °C in the range of 190–250 nm using 1 nm bandwidth in 1 mm path length quartz cuvette. CD experiments were performed only for the apo protein (HEL) at concentration of 15 μM in the pH range 4–2. Far-UV CD of HEL–CTC complexes were not analyzed due to their large signal fluctuations in the presence of imidazole derivatives. The near-UV CD experiments were performed on at 25 °C in the range of 250–340 nm using 1 nm bandwidth in 10 mm path length quartz cuvette with 50 μM of HEL.

Fluorescence spectroscopy

All the fluorescence experiments for apo HEL and with compounds were measured in the pH range 4–2 at 25 °C on Fluorolog (HORIBA JOBIN YVON) Spectrofluorometer using 4 mm path length quartz cuvette with 2.5 nm excitation slit and 2.5 nm emission slit at room temperature. Fluorescence spectra were obtained by excitation of intrinsic fluorophore tryptophan at 295 nm and the emission spectra were recorded from 300–450 nm. All the fluorescence measurements were performed in triplicate to ensure the reproducibility of the data.

Nuclear magnetic resonance (NMR) spectroscopy

1D-¹H NMR spectra of HEL samples were recorded on the Bruker Avance 500 MHz NMR spectrometer. NMR spectra recorded at the concentration of 500 μM in 100 mM glycine buffer containing 10% D₂O at pH 4–2 at 25 °C with 512 scans, and processed using Topspin 3.2 software.

Differential scanning calorimetry

HEL samples were prepared by diluting the protein to 0.2 mg ml⁻¹ followed by degassing them using GE MicroCal ThermoVac degasser. The dialysis buffer was used as control in the reference cell, and the pH values of all the samples/buffers were reconfirmed before carrying out the DSC experiment. All the experiments were performed in triplicate with each ligand to ensure the reproducibility of the data.

All the DSC experiments of HEL were performed on a VP-DSC instrument (GE MicroCal), at a scan rate of 1 °C min⁻¹, scan range from starting temperature 10 °C to ending temperature 90 °C and with the feedback mode ‘none’ and all other parameters are default. The DSC raw data were analyzed by using Origin software. The reference data were subtracted from the protein melting curve and the thermodynamic parameters were first determined by analyzing individual apparent heat capacity curves with a two-state model using a non-linear least square fitting method. The results were used as initial values for determining the thermodynamic parameters by a global fitting routine using the following equations as described elsewhere.²⁷

ΔH(T) = ΔH(T_m) + ΔC_p(T − T_m) (1)

ΔS(T_m) = ΔH_m/T (2)

Structural analysis of HEL

For structural analysis we have used HEL crystal structure PDB ID: 193L. Accessible surface areas were calculated using the GETAREA server,²⁸ and the pK_a values were calculated using the Rosetta pK_a calculation server ROSIE.²⁹ Distances between the side chain of negatively charged residue and surrounding residues were calculated using the distance measurement wizard of PyMol software.

Results

The stability of protein is indeed dependent on its structure at the given environmental conditions. Hence, we analyzed the structural modulations of the HEL in the range of pH 4 to 2 using various biophysical techniques such as CD (both far-UV and near-UV), 1D-NMR and fluorescence spectroscopy. We then correlated this structural information of HEL with its stability characteristics under identical experimental conditions. Further, we analyzed the pH dependent stability aspects of HEL in the presence of NaCl and imidazole derivatives in order to delineate their destabilizing potencies in modulating HEL stability through weak electrostatic interactions.

pH dependent structural changes in HEL

Circular dichroism is a very sensitive technique to probe the global structural changes (both secondary and tertiary) in the proteins. Far-UV CD in the wavelength range 190–240 nm furnishes information about the changes in secondary structural elements of protein, whereas near-UV CD in the wavelength range 250–320 nm render the changes regarding tertiary structure. In order to assess the secondary and tertiary structural changes, we have performed CD experiments on HEL (Fig. 2A and B). CD results indicated that, there is no observable change in the far-UV CD spectral pattern at the measured pH values, establishing that HEL has identical secondary content at all the pH values (Fig. 2A). Further we probed the authenticity and localization of these local tertiary changes in the vicinity of aromatic residues using the near-UV CD (Fig. 2B). Tertiary CD clearly evidenced for a variation in the tertiary structure features of HEL at measured pH as the features of pH 2 is significantly different from those of the pH 4 and 3 conformation, which are closer to each other. Further, to substantiate this observation, we have performed the fluorescence spectroscopy using intrinsic tryptophan residues of HEL, as Trp fluorescence is a very sensitive monitor of such local changes. HEL comprises of six tryptophan residues and the observed fluorescence is an average of emission of all these fluorophores. In the fluorescence spectra, we do observe some change in the overall intensity profile upon reducing pH. As assessed earlier, HEL at pH 4 and 3 have very similar conformation but not at pH 2 as evidenced for an overall 15% hypochromic shift (Fig. 2C). The hypochromic behavior of fluorescence intensity without any bathochromic shift reflects the fact that, during this pH transition the buried Trp residues have potentially reoriented/exposed their aromatic rings with respect to the overall charge/polarity of the solvent hydration/environment. In order to confirm the observed pH dependent tertiary/local structural changes are indeed significant, we have performed 1D ¹H NMR experiments (Fig. 2D). Proton NMR spectroscopy acts as a fingerprint in monitoring the protein structural changes. Overlay of HEL 1D ¹H NMR spectra at pH 4 (black), pH 3 (blue) and pH 2 (red) evidenced some minor changes in the peak intensities and chemical shifts at both the NH and the CH spectral region at pH 2 in comparison to that of pH 3 and pH 4, which are more or less similar, thus accounting for the local structural changes in the tertiary interactions/side chain packing/orientations of HEL. All these results indicated that, although the global/secondary structural composition of HEL is same at all the pH values 4–2, the local tertiary structural/side chain interactions do differ noticeably.

Fig. 2 Spectroscopic measurements on HEL at 25 °C in the acidic pH – pH 4.0 (black), pH 3.0 (blue) and pH 2.0 (red); (A) far-UV CD profile showing the secondary structural changes; (B) near-UV CD profile showing the tertiary structural changes; (C) tryptophan fluorescence emission spectra; (D) overlay of NH-region (left panel) and CH-region (right panel) of 1D ¹H-NMR spectra.

Differential stability features of HEL

To decipher the associated thermodynamic characteristics of HEL in the range of pH 4 to 2, we have performed the thermal denaturation experiments using differential scanning calorimetry (DSC) (Fig. 3A). DSC is a very sensitive technique to monitor the thermal stability of the proteins.^27,30–32 In DSC, one can obtain all the thermodynamic parameters in single scan with great accuracy.³³ Literature reports on HEL denaturation profiles suggest that, the protein denaturation follows a two state transition, in which only the native and denatured states are significantly populated.³⁴ The pH dependent denaturation profiles of the HEL were analyzed considering the two state model, where the van't Hoff enthalpy was calculated using the van't Hoff transformations.³⁵ The obtained ratio of calculated enthalpy change (ΔH_cal) and van't Hoff enthalpy change (ΔH_v) is consistently close to 1, suggesting the correctness of the approximation (Table 1).^36,37

Fig. 3 Thermodynamic characterization of apo HEL at low pH using differential scanning calorimetry; (A) DSC thermogram of apo HEL at pH 4.0 (black), pH 3.0 (blue) and pH 2.0 (red); (B) specific enthalpy difference (ΔH) curve; (C) specific entropy difference (ΔS) profile; (D) temperature dependent Gibbs free energy curves in the temperature range 15–90 °C during the denaturation of the HEL.

Table 1 Thermodynamic parameters obtained for HEL at pH 4, pH 3 and pH 2 using the DSC denaturation curves in the presence of different compounds

pH	Compound	Tm (°C)	ΔH (cal mol^−1)	ΔHv (cal mol^−1)	ΔH/ΔHv
pH 4.0	Apo	77.3	120 298 ± 425	122 800 ± 231	0.98
	Imidazole	73.2	129 755 ± 175	124 500 ± 185	1.04
	Histidine	71.5	107 597 ± 234	111 230 ± 278	0.97
	Histamine	72.2	112 534 ± 243	109 536 ± 352	1.09
	NaCl	73.3	118 000 ± 837	110 000 ± 923	1.07
pH 3.0	Apo	67.5	128 623 ± 400	113 100 ± 218	1.13
	Imidazole	60.8	109 880 ± 350	108 400 ± 277	1.01
	Histidine	62.4	115 462 ± 165	101 800 ± 183	1.13
	Histamine	60.7	126 814 ± 270	119 900 ± 282	1.06
	NaCl	61.8	108 900 ± 464	104 000 ± 549	1.05
pH 2.0	Apo	47.6	84 000 ± 145	78 690 ± 165	1.06
	Imidazole	47.6	85 224 ± 308	79 630 ± 207	1.07
	Histidine	48.0	89 574 ± 205	84 600 ± 235	1.06
	Histamine	47.9	77 883 ± 285	71 470 ± 350	1.08
	NaCl	47.7	83 765 ± 234	81 647 ± 354	1.03

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DSC denaturation data shows that melting temperature (T_m) decreases with the decreasing the pH toward the acidic pH (Table 1), evidencing the thermal fragility of the HEL at acidic pH. Stability of HEL at pH 2 (T_m = 47.6) is much lower compared to its pH 3 (T_m = 67.5) and pH 4 (T_m = 77.3) conformation, although both states demonstrated similar secondary structural characteristics in the spectroscopic studies as discussed above. The decrease of melting points upon decrease of pH can be contributed to the observed differential tertiary interactions of HEL. Further, we observed that the thermogram at pH 2 is much broader compared to pH 3/4, thus representing the destabilization of the native state in a differential way by modulating its thermodynamic parameters, which is an interplay of enthalpy, entropy and the heat capacities of the transition (Fig. 3A). To gain more insights into the thermodynamic parameters obtained from these pH dependent thermal denaturation experiments, we have analyzed the individual components such as enthalpy changes ΔH(T), entropy changes ΔS(T), and free energy ΔG(T), by using the thermodynamic equations as described in materials and methods (Fig. 3B–D). Thermodynamic profiles clearly represent the lower stability of HEL at pH 2 as free energy values are less by ∼5 kcal mol⁻¹ in equivalence to pH 3 and pH 4, which contain similar ΔG values at 25 °C (Fig. 3D). From all the above thermodynamic, it is evident that upon lowering the pH of HEL from 4 to 2, a significant amount of stabilization free energy has been sacrificed.

Monitoring the HEL structural changes upon interaction with imidazole derivatives

The fluorescence spectroscopy had been reported as one the best technique to monitor the formation of charge transfer complexes between the tryptophan residues of protein and the positively charged imidazole and its derivatives.³⁸ At low pH values CTCs are reported to form between the exposed Trp residues of HEL with several imidazole like compounds. Further these complexes alter the HEL catalytic activity,^10,23,39 as the CTC formation involves the Trp residue (W62), present at its active site.^21,40 In order to confirm the interaction between the positively charged imidazole derivatives such as imidazole, histidine, and histamine (used in this study Fig. 1C) with the tryptophan residues of the HEL, we have performed fluorescence experiments with these compounds, by monitoring the intrinsic tryptophan fluorescence of HEL (Fig. 4A–C). The fluorescence spectra in Fig. 4 clearly manifested the quenching of HEL tryptophan fluorescence upon addition of imidazole derivatives to HEL, thus indicating the formation of the charge transfer complex at all the measured pH values (pH 4–2). Furthermore, we noticed that the percentage of the fluorescence quenching by the imidazole derivatives differs, and the intensities are attenuated around 15–30%. Moreover, the pattern of quenching is similar in the measured pH range for the compound under consideration, establishing that HEL forms similar CTC complexes at all pH values with these imidazole derivatives. Imidazole being the smallest of all the three derivatives quenched the fluorescence by a maximum of ∼25–30% in comparison to the rest of the derivatives histidine and histamine (∼15%). Further, to substantiate the fact that such an interaction observed is not due to just electrostatic nature, we have obtained the far-UV and fluorescence spectra of HEL in the presence of NaCl (Fig. S1, ESI†). Addition of NaCl does not alter the CD profiles and fluorescence intensities of the HEL suggesting that the observed changes are indeed due to the specific interactions with Trp residues in the form of CTC's, which is in line with the literature reported earlier.

Fig. 4 In the figure, left panel (A–C) represents the changes in the fluorescence emission spectra of HEL at (A) pH 4.0 (black), (B) pH 3.0 (blue) and (C) pH 2.0 (red), and in complex with imidazole (green), histidine (pink) and histamine (cyan). The right panel (D–F) represents the near-UV CD profile of HEL in apo and with the same compounds as represented in left panel. The changes observed in near-UV CD upon addition of NaCl (dark red) are also represented.

As, the compounds carry positive charge, certain extent of modulation of structural features were expected upon addition of these compounds to HEL. To monitor those structural changes, we have performed both far-UV and near-UV experiments with the imidazole derivatives and NaCl as a reference for electrostatic interactions. Far-UV with NaCl does not alter the structural features of HEL (Fig. S1, ESI†). We could not measure reliable far-UV profiles of HEL in the presence of imidazole derivatives due to large fluctuations in the signals. We then measured the near-UV features with all these compounds and the observed profiles are shown in Fig. 4D–F. In the case of pH 4 and pH 3 there is a systematic change of CD-signals in the presence of these compounds and also with reference compound NaCl, whereas the changes are less and random at pH 2 suggesting that these compounds influence the structure of HEL via electrostatic interactions and also in a pH dependent manner.

Effect of electrostatic interactions on the thermodynamic stability of the HEL

Our intent of the study is to explore the stability aspects of HEL in the presence of compounds that interact electrostatically upon formation of the CTCs complexes. As we have observed the formation of CTC complexes for all the titrated compounds in the pH range 4–2, we have performed DSC experiments on HEL–CTC complexes with all these imidazole derivatives. The results of the thermograms and the stability parameters set are presented in Fig. 5, Table 1. As a reference to electrostatic interactions, we have performed the DSC experiments in the presence of same concentration of NaCl. Analysis of the DSC experiments has depicted some interesting and novel features regarding the thermodynamic aspects of HEL–CTC complexes: (i) at all the pH values (4–2), the denaturation profile follows a simple two state transition, which is evident from the ratio of ΔH/ΔH_v values (Table 1). (ii) The electrostatic interactions of the CTC forming imidazole derivatives resulted in pH dependent differential stability features of HEL. At pH 4 and pH 3, these interactions resulted in a considerable decrease (5–7 °C) in HEL's melting temperature (Fig. 5A and B, Table 1). The stability behavior of histamine and imidazole with HEL are very similar with a decrease of T_m ∼ 7 °C, and showed a marginal destabilizing effect compared to histidine and the reference compound NaCl. However, surprisingly in the case of pH 2, the thermal denaturation profiles of the HEL are untouched although the formation of CTCs is very similar to those at high pH values (Fig. 4A–C, 5C and Table 1). Such a differential features in the melting transitions at the measured pH values indicate that the structural interactions of these compounds at pH 2 are far different to those at pH 3 and 4.

Fig. 5 DSC thermograms showing the denaturation profile of HEL at (A) pH 4.0 (black), (B) pH 3.0 (blue), and (C) pH 2.0 (red) in complex with imidazole (green), histidine (pink), histamine (cyan) and NaCl (dark red).

Discussion

Mechanistic insights into pH dependent differential stability attenuation of HEL upon interaction with imidazole derivatives

Electrostatic contacts between the positively charged amino acids with negative charged and aromatic ring side chains are often found in proteins.^41,42 Involvement of the imidazole and its derivatives with the tryptophan in the charge transfer type complexes has intrigued biological and biophysical chemists to unravel their mechanistic role in the biomolecular systems as imidazole ring is present in the various types of biological compounds including bases of nucleotides, vitamin B12, histamine, histidine and some natural and synthetic drug molecules.⁴³

In the same context we have studied the role of electrostatic contributions on the stability profile of HEL using CTC forming imidazole derivatives in a pH dependent manner. Fluorescence experiments established that the imidazole derivatives do form CTC's with the HEL. The low pH conformation of HEL possesses similar secondary structural elements at all the measured pH values (pH 4 to 2) and a certain degree of variation is observed in the tertiary interactions as evidenced by NMR and near-UV CD. Our results suggested that the intrinsic stability of the low pH conformation of HEL decreases drastically i.e., ∼20 °C with decrease of pH from 4 to 2 (Fig. 3A, Table 1). Such a decrease of stability can be directly attributed to the ionizable groups present in the protein in that pH range. HEL has two Glu residues and seven Asp residues, whose pK_a values are spanned in the range of 2.5–4.0 with varying solvent accessibility (Fig. 6A and B, Table 2). Moreover, several of these Glu/Asp residues are buried inside the protein and are making contacts with several other hydrophobic, aromatic and hydrophilic side chains of the protein core. Upon decreasing the pH, these negatively charged Asp and Glu residues are protonated, facilitating the perturbation of the network of native HEL side chain interactions, thus varying the tertiary structural interactions and decreasing its stability (Fig. 6C, Table 2).

Fig. 6 (A) Cartoon representation of the HEL structure with all its Glu and Asp (acidic amino acids) side chains highlighted. (B) The surface representation of acidic residues (Glu and Asp) from both sides by using a 180° rotation, (C) the side chain atomic distances (<6 Å) between the interacting residues with those of E35, D48 and D66 in the HEL structure (PDB ID: 193L). The figure was generated using PyMol molecular graphics software.

Table 2 Summary of the pK_a values, accessible surface areas and side chain interactions of acidic ionizable groups (Glu, Asp) in HEL

Residue no.	pKa	Accessible surface area		Interacting residues (residue name (atom name, distance Å))
		Total accessible surface area (%)	Side chain surface area (A)^2
Glu7	3.6	58.1	82.10	K1(NZ, 2.8), F3(CG, 4.1)
Asp18	3.2	30	33.92	N19(ND2, 3.0), L25(CD1, 5.5)
Glu35	3.8	13.6	19.27	Q57(CB, 3.3), W108(CD1, 3.8), V109(CG1, 4.0), A110(CB, 3.4)
Asp48	2.4	48.4	54.65	N46(OD1, 5.5), S50(OG, 2.7), N59(ND2, 5.1), R61(NH2, 3.4)
Asp52	4.1	22.1	24.95	N44(CB, 4.9), N46(ND2, 4.1), Q57(OE1, 4.3), N59(ND2, 2.8)
Asp66	2.5	7.2	8.11	R45(NH2, 6.4), Y53(OH, 2.8), S60(OG, 3.3), R68(CG, 3.8), T69(OG1, 3.3)
Asp87	2.9	66.5	75.15	R14(NH2, 6.0), H15(NE2, 4.4), T89(OG1, 2.6)
Asp101	3.8	47	53.12	W63(CH2, 3.9), L75(CD1, 5.5), N103(ND2, 4.5)
Asp119	3.5	76.2	86.11	A122(CB, 5.0)

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In a similar note, one can explain the observed differential interacting behavior of the imidazole derivatives and NaCl over HEL stability. The pK_a of the Glu/Asp residues in the range of 2.5–4.0 suggests that, several of these residues exist in mixed populations of both protonated and unprotonated states. These mixed populations of HEL protonated species is the primary source of the structural instability. The interaction of the positive imidazole ions with the populations of the unprotonated Asp/Glu side chains at pH 4 and 3 contributes to the further decrease of the stability around 5–7 °C, depending on the individual charge distributions of the compounds. However, at pH 2, almost all the side chains in the HEL conformation has completely protonated and destabilized several of those protein's interaction networks. In such a scenario further addition of positively charged compounds does not influence the structural stability as no more destabilizing interactions are plausible, thus leaving the conformation of pH 2 untouched. Our results apparently demonstrated that, the ultra-weak electrostatic interactions of the protein molecule with the surrounding solute/interacting molecules can potentially influence the native stability of the protein. These results provided mechanistic insights into the origin of pH dependent stability attenuation of imidazole derivatives and the regulatory roles of non-covalent electrostatic interactions in maintaining or modulating the conformational stability of proteins.

Concluding remarks

In summary, we have delineated the pH dependent stability characteristics of electrostatic interactions between the protonated forms of imidazole derivatives with HEL at acidic pH. Our studies established that these charged interactions between the compounds and the exposed unprotonated Glu/Asp side chains of the protein potentially attenuate its conformational stability. Furthermore, these energetic changes are highly dependent on the pH as the percent of the net protonation of an amino acid (pK_a) depends on the pH of solvent and the extent of solvent accessibility. Likely, in the case of HEL, a group of solvent exposed and partially buried Asp and Glu residues differentially contribute to these electrostatic interactions and hence mediated the stability features. The mechanistic details elucidated here have provided novel insights into the structure–stability paradigm of proteins involving non-covalent and weak electrostatic interactions and will aid us in translating our knowledge to decode the complex networking principles of biomolecular interactions.

Author contributions

KMP designed the research, KG performed the experiments, KG, KMP analyzed the data and wrote the manuscript.

Abbreviations

HEL	Hen egg lysozyme
NMR	Nuclear magnetic resonance
DSC	Differential scanning calorimetry
CTC	Charge transfer complex
CD	Circular dichroism

Acknowledgements

This work is supported by the DBT-IYBA fellowship – BT/07/IYBA/2013-19, SERB-SB/YS/LS-380/2013 and startup aid from MHRD-IITR, Government of India (GoI) to KMP. Authors acknowledge the support of NMR Instrumentation center at IIT-Roorkee for the access of NMR spectrometer and other biophysical techniques. Authors thank Prof. Michael Williamson, University of Sheffield, UK for thought provoking discussions; Ms Khushboo Gulati and Ms Meenakshi Sharma, IIT-Roorkee for the technical help.

References

1. K. A. Dill and H. S. Chan, Nat. Struct. Biol., 1997, 4, 10–19.
2. V. Muñoz and L. Serrano, Folding Des., 1996, 1, R71–R77.
3. P. L. Privalov and G. I. Makhatadze, J. Mol. Biol., 1992, 224, 715–723.
4. E. R. Johnson, S. Keinan, P. Mori-Sanchez, J. Contreras-Garcia, A. J. Cohen and W. Yang, J. Am. Chem. Soc., 2010, 132, 6498–6506.
5. S. Burley and G. Petsko, Adv. Protein Chem., 1988, 39, 125–189.
6. E. Frieden, J. Chem. Educ., 1975, 52, 754–761.
7. J. Černý and P. Hobza, Phys. Chem. Chem. Phys., 2007, 9, 5291–5303.
8. M. Inoue, M. Shibata, Y. Kondo and T. Ishida, Biochemistry, 1981, 20, 2936–2945.
9. S. Mecozzi, A. P. West and D. A. Dougherty, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 10566–10571.
10. M. Shinitzky, E. Katchalski, V. Grisaro and N. Sharon, Arch. Biochem. Biophys., 1966, 116, 332–343.
11. M. Shinttzky and R. Goldman, Eur. J. Biochem., 1967, 3, 139–144.
12. M. Shinitzky and M. Fridkin, Eur. J. Biochem., 1969, 9, 176–181.
13. M. Shopova and N. Genov, Int. J. Pept. Protein Res., 1983, 21, 475–478.
14. D. Deckers, D. Vanlint, L. Callewaert, A. Aertsen and C. W. Michiels, Appl. Environ. Microbiol., 2008, 74, 4434–4439.
15. A. Miranker, S. E. Radford, M. Karplus and C. M. Dobson, Nature, 1991, 349, 633–636.
16. D. M. Rothwarf and H. A. Scheraga, Biochemistry, 1996, 35, 13797–13807.
17. D. C. Phillips, Proc. Natl. Acad. Sci. U. S. A., 1967, 57, 483–495.
18. C. C. Blake, G. Mair, A. North, D. Phillips and V. Sarma, Proc. R. Soc. London, Ser. B, 1967, 167, 365–377.
19. M. Buck, J. Boyd, C. Redfield, D. A. MacKenzie, D. J. Jeenes, D. B. Archer and C. M. Dobson, Biochemistry, 1995, 34, 4041–4055.
20. D. J. Vocadlo, G. J. Davies, R. Laine and S. G. Withers, Nature, 2001, 412, 835–838.
21. C. Rmoso and L. S. Forster, J. Biol. Chem., 1975, 250, 3738–3745.
22. T. Imoto, L. S. Forster, J. Rupley and F. Tanaka, Proc. Natl. Acad. Sci. U. S. A., 1972, 69, 1151–1155.
23. I. D. Swan, J. Mol. Biol., 1972, 65, 59–62.
24. M. Stuart-Audette, Y. Blouquit, M. Faraggi, C. Sicard-Roselli, C. Houée-Levin and P. Jolles, Eur. J. Biochem., 2003, 270, 3565–3571.
25. M. Weinstein, Z. B. Alfassi, M. R. DeFelippis, M. H. Klapper and M. Faraggi, Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol., 1991, 1076, 173–178.
26. L. M. Hinman, C. R. Coan and D. A. Deranleau, J. Am. Chem. Soc., 1974, 96, 7067–7073.
27. A. Kato, M. Yamada, S. Nakamura, S.-i. Kidokoro and Y. Kuroda, J. Mol. Biol., 2007, 372, 737–746.
28. R. Fraczkiewicz and W. Braun, J. Comput. Chem., 1998, 19, 319–333.
29. K. P. Kilambi and J. J. Gray, Biophys. J., 2012, 103, 587–595.
30. G. Bruylants, J. Wouters and C. Michaux, Curr. Med. Chem., 2005, 12, 2011–2020.
31. M. Cueto, M. J. Dorta and O. Munguí, Int. J. Pharm., 2003, 252, 159–166.
32. J. W. Donovan, C. J. Mapes, J. G. Davis and J. A. Garibaldi, J. Sci. Food Agric., 1975, 26, 73–83.
33. A. D. Robertson and K. P. Murphy, Chem. Rev., 1997, 97, 1251–1268.
34. F. Meersman, C. Atilgan, A. J. Miles, R. Bader, W. Shang, A. Matagne, B. A. Wallace and M. H. Koch, Biophys. J., 2010, 99, 2255–2263.
35. F. Delben and V. Crescenzi, Biochim. Biophys. Acta, Protein Struct., 1969, 194, 615–618.
36. S. I. Kidokoro, H. Uedaira and A. Wada, Biopolymers, 1988, 27, 271–297.
37. S. I. Kidokoro and A. Wada, Biopolymers, 1987, 26, 213–229.
38. J. Jayabharathi, V. Thanikachalam and M. V. Perumal, J. Lumin., 2012, 132, 707–712.
39. R. Field, A. H. Haines, E. J. Chrystal and M. C. Luszniak, Biochem. J., 1991, 274, 885–889.
40. T. Imoto, L. Johnson, A. North, D. Phillips and J. Rupley, The enzymes, 1972, vol. 7, pp. 665–868.
41. J. Fernández-Recio, A. Vázquez, C. Civera, P. Sevilla and J. Sancho, J. Mol. Biol., 1997, 267, 184–197.
42. D. A. Dougherty, Science, 1996, 271, 163–168.
43. A. Verma, S. Joshi and D. Singh, J. Chem., 2013, 2013, 1–12.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra23031h

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