- Email: [email protected]
Local pH at the surface of hen egg white lysozyme
Accepted Manuscript Research paper Local pH at the surface of hen egg white lysozyme Takuhiro Otosu, Kaito Kobayashi, Shoichi Yamaguchi PII: DOI: Reference:
S0009-2614(18)30034-4 https://doi.org/10.1016/j.cplett.2018.01.026 CPLETT 35378
To appear in:
Chemical Physics Letters
Received Date: Accepted Date:
19 December 2017 10 January 2018
Please cite this article as: T. Otosu, K. Kobayashi, S. Yamaguchi, Local pH at the surface of hen egg white lysozyme, Chemical Physics Letters (2018), doi: https://doi.org/10.1016/j.cplett.2018.01.026
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Local pH at the surface of hen egg white lysozyme
Takuhiro Otosu, Kaito Kobayashi, and Shoichi Yamaguchi* Department of Applied Chemistry, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura, Saitama 338-8570, Japan *[email protected]
Keywords: lysozyme, fluorescein, pH at the protein surface
1
Abstract The microenvironment at the surface of hen-egg-white lysozyme (HEWL) was examined by analyzing the change in pKa of fluorescein isothiocyanate (FITC) upon binding to the N-terminus of HEWL. The result showed that the local pH at the HEWL surface is higher than the bulk pH. Furthermore, the data showed that the difference between the local and bulk pH becomes larger with decreasing pH, suggesting HEWL repels more protons at lower pH. Because the local pH affects the protonation states of functional amino-acids at the protein surface, the results provide the fundamental insight into the microenvironment at the protein surface.
Highlights Local pH at the surface of hen-egg-white lysozyme (HEWL) was examined. The local pH is higher than the bulk pH due to the net positive charge of HEWL. The pH difference becomes larger with decreasing the bulk pH.
2
1. Introduction Various kinds of biological functions (e.g., signal transduction, replication and duplication of DNA, and respiration) achieved or mediated by proteins are initiated with the interaction between the protein surface and its target molecules. Attractive forces between these molecules due to electrostatic and/or hydrophobic interactions promote the effective formation of the protein-target complex [1]. On the other hand, strong attractive interaction causes the aggregate formation which leads to fatal diseases such as Alzheimer’s disease and Parkinson disease [2-4]. Therefore, various spectroscopic analyses have been attempted to elucidate the chemical properties of the microenvironment at the protein surface for understanding the sophisticated biological functions expressed by proteins. For example, steady-state absorption as well as fluorescence spectroscopies of tryptophan residue or some extrinsic probes have revealed the local polarities at the protein surface [5-7]. In addition to the static properties at the protein surface, time-resolved fluorescence spectral shift of tryptophan, which reflects the reorganization of water molecules surrounding the probe, has shown the dynamic nature of the microenvironment at the protein surface [8-12]. Vast of such studies have revealed that the dynamics of water at the protein surface is much slower than that of bulk water, which is often described as “biological” water [10,11]. Because biological water is expected to be excluded from the protein surface during the formation of protein-target complex, the reorganization dynamics of those water molecules has the fundamental insight into the kinetics of protein-target complex formation. Therefore, previous spectroscopic studies have been focused on the static and dynamic properties of the microenvironment at the protein surface. However, the electrostatic property of the protein surface, that is, local pH at the surface has not been extensively studied
except
for
bacteriorhodopsin
[13,14].
Because
the
local
pH
determines
the
protonation/deprotonation states of amino acids and target molecules located at the protein surface, estimation of this quantity is crucial to understand the various biological functions of proteins. In this letter, we analyzed the local pH at the surface of hen-egg-white lysozyme (HEWL) 3
Fig.
1.
X-ray
crystallographic
structure
of
hen-egg-white lysozyme (PDB ID: 3RZ4). Lysine residue
at
the
N-terminus,
where
fluorescein
isothiocyanate is bound, is shown by a stick.
through the change in the negative logarithm of the acid dissociation constant, pKa, of fluorescein isothiocyanate (FITC) upon covalent binding to the N-terminus of HEWL. HEWL is a small water-soluble protein which has been used as a model protein for protein folding, crystallization, and so on (Fig. 1) [15-18]. One remarkable feature of HEWL is its conformational stability against pH [18], which makes HEWL a suitable protein for analyzing the local pH at the surface of a folded protein via pH dependence of the absorption spectra of FITC bound to HEWL (hereafter referred to as FITC-Lz).
2. Materials and Methods Hen-egg-white lysozyme (HEWL) was purchased from Sigma-Aldrich, papain was purchased from Wako, and fluorescein isothiocyanate (FITC) was purchased from Dojindo. All reagents used in this study were of analytical grade and used without purification. To label HEWL with FITC, lyophilized powder of HEWL was dissolved into 20 mM sodium phosphate buffer (pH 7.5) containing 100 mM NaCl. The HEWL solution (~100 M) was then mixed with FITC solution (~50 4
mM) in dimethyl sulfoxide with a molar ratio of 1:2 and stirred for 2 h at room temperature. In this pH condition, FITC is expected to be selectively bound to the N-terminal -amine of HEWL [16]. To remove the unreacted FITC, the mixed solution was applied to PD-10 desalting column (GE Healthcare Life Sciences) that was equilibrated with 100 mM NaCl in water in advance. The labeling efficiency of FITC was estimated from the absorption spectrum of the sample and the extinction coefficient of HEWL (37800 M-1 cm-1 at 280 nm) and FITC (64800 M-1 cm-1 at 494 nm and 20400 M-1 cm-1 at 280 nm), and it was ~0.3 [16,19]. The FITC labeling of papain was performed with the same procedure as HEWL, and the labeling efficiency was ~0.4. For the absorption measurements, FITC-labeled proteins of ~20 M were diluted four times by using the following buffers depending on pH: 100 mM sodium phosphate buffer for pH 9.0–5.5, 100 mM sodium acetate buffer for pH 5.0–3.0, both of which contain 100 mM NaCl. Absorption spectra of those samples were then measured at 350–550 nm with the increment of 0.5 nm by using a UV-vis spectrophotometer (U-2900, HITACHI). Because the sample was limited and the protein might adsorb on the glass electrode of a pH meter (KR5EW, AS ONE), it was difficult to directly measure the bulk pH of the sample. Thus, the sample solution in the absence of labeled proteins was prepared by mixing 100 mM NaCl in water with the dilution buffer at the same mixing ratio as the sample and was used for the pH measurement to estimate the bulk pH of the sample. For the measurements of FITC in 7 vol% acetone, 20M FITC of 500 L in water containing 128 mM NaCl was mixed with the dilution buffer of 1360 L mentioned above and acetone of 140L. The bulk pH of the sample was also independently measured by preparing the same solution without FITC.
3. Results and Discussion Figure 2(a) shows the absorption spectra of FITC-Lz at various bulk pHs. At pH ~9.0, the spectrum shows the absorption maximum at ~490 nm with a smaller shoulder at ~455 nm. When pH 5
Fig. 2. (a) Absorption spectra of fluorescein isothiocyanate on the N-terminus of hen-egg-white lysozyme at various pHs. The bulk pH of each spectrum is shown in the figure. The data were fitted with Eqs. (1)-(4) and the reconstructed spectra are shown in (b).
is changed to pH 5.0, the band at ~490 nm decreases and is blue shifted to ~480 nm. Furthermore, a new peak at ~450 nm emerges. The absorbance at ~480 nm and ~450 nm decreases by further decreasing pH to ~3.0, and the latter peak is blue shifted to ~440 nm. Therefore, pH dependence of the absorption spectra of FITC-Lz suggests that three species with different absorption spectra are involved in the spectrum as reported for free fluorescein in this pH range [20,21]. To determine the pKa value of FITC-Lz, pH dependence of the absorption spectra shown in Fig. 2(a) was analyzed. At the pH range from 3 to 9, FITC can be regarded as a diprotic acid in successive equilibria [20,21]: H2A ⇄ H+ + HA- ⇄ 2H+ + A2-.
- scheme 1
Therefore, the absorption spectrum of the sample (A(pH, λ)) can be described with the theoretical equation for three-state equilibria as follows:
ApH,
AH 2A ( ) 10 pHpKa1 AHA ( ) 10 2 pHpKa1 pKa2 AA 2- ( ) 1 10 pHpKa1 10 2 pHpKa1 pKa2
.
(1)
In Eq. (1), pKa1 and pKa2 are the pKas between H2A and HA- and between HA- and A2-, respectively, and AH 2 A ( ) , AHA- ( ) , and AA 2- ( ) are the absorption spectra of H2A, HA- and A2-, respectively. 6
The absorption spectrum of each species can be described with a linear combination of experimentally obtained spectra at arbitrary pHs as follows: AH 2A ( ) 1 A(pH1 , ) 2 A(pH2 , ) 3 A(pH3 , ) ,
(2)
AHA- ( ) 1 A(pH1 , ) 2 A(pH2 , ) 3 A(pH3 , ) ,
(3)
AA 2- ( ) 1 A(pH1 , ) 2 A(pH2 , ) 3 A(pH3 , ) .
(4)
In the actual analysis, pH1, pH2, and pH3 were set to 3.1, 6.1, and 8.9, respectively. All spectra obtained at different pHs were then globally fitted by using Eqs. (1)-(4), and the fitting parameters pKa1, pKa2, i, i, and i (i = 1-3) were determined. Figure 2(b) shows the reconstructed spectra of FITC-Lz that were obtained by the global fitting of the experimental data with Eqs. (1)-(4). (The absorption spectra of H2A, HA- and A2- are shown in Fig. S1.) Agreement between the experimental and reconstructed spectra confirms that the three-state model (scheme 1) is adequate to reproduce the essential features of the spectral change such as the peak shift and the existence of the isosbestic point at ~460 nm. Based on the fitting, pKa1 and pKa2 were determined to be 3.79 and 6.31, respectively. It is important to note that the obtained pKa is the “apparent” one because the data shown in Fig. 2(a) were analyzed by using the bulk pH that is not necessarily the same as the pH that the probe experiences. Actually, recent sum-frequency generation studies of the charged surfaces have shown that the local pH at the charged surface is different from the bulk pH due to the electrostatic repulsion/attraction of protons from/to the surface [22,23]. Indeed, the isoelectric point (pI, the pH where the net charge becomes zero) of HEWL is 11.35 and HEWL is positively charged at the pH range examined in this study [24]. Thus, the local pH at the HEWL surface is anticipated to be higher than the bulk pH. While the apparent pKa was determined with respect to the bulk pH, the “intrinsic” pKa of the probe at the protein surface is defined with respect to the local pH. The intrinsic pKa is thus described using the apparent one as follows, taking account of the difference (ΔpH) between the 7
local pH and the bulk pH:
Fig. 3. (a) Absorption spectra of fluorescein isothiocyanate (FITC) in different water-acetone mixture at pH 9.0. For comparison, the spectrum of FITC on hen-egg-white lysozyme (HEWL) measured at pH 8.9 is also shown (black solid line). Data are normalized to match the absorption maximum. (b) Relationship among the absorption peak wavelength of FITC, the dielectric constant of the solvent, and the volume percentage of acetone in water-acetone mixture. Data (open circle) were obtained from (a), and fitted with a linear function (red broken line). Peak wavelength of FITC on HEWL at pH 8.9 is also plotted on the fitting data (black rectangle).
intrinsic pKa = apparent pKa + local pH – bulk pH = apparent pKa + ΔpH.
(5)
Therefore, the evaluation of intrinsic pKa is needed to get the information of the local pH. Because pKa is given by the standard reaction Gibbs energy from the acid to the base, intrinsic pKa is directly related to the local effective polarity (or dielectric constant) of medium surrounding the probe [25]. Thus, the effective dielectric constant at the probe position was first examined by measuring the solvatochromic shift of the FITC absorption spectrum upon binding to the HEWL surface. Figure 3(a) shows the absorption spectra of free FITC in solution containing different vol% of acetone. The samples were prepared by mixing FITC in sodium phosphate buffer at pH 9.0 with acetone. At this pH, A2- is a dominant species in FITC. The spectrum of A2- is shifted to longer wavelength by increasing the mixing ratio of acetone (decreasing the dielectric constant of the solvent), which indicates negative solvatochromism. Therefore, this result suggests that the peak wavelength of A2- is a good indicator to estimate the effective dielectric constant at the protein 8
Fig.
4.
Absorption
spectra
of
fluorescein
isothiocyanate in 7 vol% acetone at various pHs. The bulk pH of each spectrum is shown in the figure. The data were fitted with Eqs. (1)-(4) and the reconstructed spectra are shown by broken lines.
surface. Figure 3(b) shows the relationship between the dielectric constant of the solvent and the peak wavelengths of FITC. The data were fitted with a linear function and the result is also shown in the figure. The dielectric constants of water-acetone mixture were obtained from literature [26]. Based on the fitting and the peak wavelength of FITC-Lz at pH ~9.0, it was found that the effective dielectric constant at the HEWL surface is equivalent to that in 7 vol% acetone in water. It is worth noting that the conformation of HEWL is robust and insensitive to pH from 3.0 to 9.0 [18]. Therefore, it is reasonable to assume that the effective dielectric constant at the HEWL surface is independent of pH. To determine the intrinsic pKa of FITC-Lz, the absorption spectra of free FITC in 7 vol% acetone at various pHs were measured. The results are shown in Fig. 4. As expected, the absorption maximum at pH 9.0 was the same as that in FITC-Lz at the same pH. Based on the fitting of the data shown in Fig. 4, pKa1 and pKa2 were determined to be 4.15 and 6.37, respectively, which correspond to the intrinsic pKas of FITC bound to HEWL. Using Eq. (5), ΔpH (= intrinsic pKa – apparent pKa) was then calculated to be 0.36 at around the bulk pH ~4 and 0.06 at around the bulk pH ~6. Positive ΔpH indicates that the local pH is higher than the bulk pH, that is, a proton is repelled from the HEWL surface. It is reasonable because the pI of HEWL is ~11.4 and thus HEWL possesses a net positive charge at the pH region measured in this study. Interestingly, the result also showed that ΔpH 9
Fig. 5. (a) Absorption spectra of fluorescein isothiocyanate on the N-terminus of papain at various pHs. The bulk pH of each spectrum is shown in the figure. The data were fitted with Eqs. (1)-(4) and the reconstructed spectra are shown in (b).
becomes larger by decreasing the bulk pH from ~6 to ~4. Because positive ΔpH arises from the proton repulsion from the HEWL surface, this pH dependency can be ascribed to the increase in the net positive charge of HEWL upon decreasing the bulk pH. In other words, the result suggests that some ionizable residues in HEWL are protonated (or neutralized from the base form) by changing the bulk pH from 6 to 4. Actually, Webb et al. analyzed the pKa of ionizable amino-acid residues in HEWL and showed that several amino acids such as His15 have pKa in between pH 6 and 4 [27]. Therefore, the change in the protonation states of such amino acids might be reflected on the pH dependence of the proton repulsion from the HEWL surface. It is tempting to think whether this observation is specific to HEWL or rather general for the protein with a high pI. Actually, the N-terminal amino acid of HEWL is a lysine residue so that the specific interaction between FITC and the amino group in the lysine residue might contribute to the ΔpH observed here. In this regard, we also analyzed the local pH at the papain surface. Papain is also a water-soluble small protein which has a pI of 9.55 and possesses an isoleucine residue on its N-terminus [28]. The absorption spectra of FITC-papain and these fitting curves with Eqs. (1)-(4) are shown in Fig. 5. The peak wavelength at pH ~9.0 was the same as that of FITC-Lz, suggesting that 10
Table 1 Fitting results and the isoelectric points (pI) of proteins
the effective dielectric constant at the probe position was equivalent to that of FITC-Lz (corresponding to 7 vol% acetone). Fitting results are summarized in Table 1. The results showed that the apparent pKas of papain are lower than the intrinsic ones (corresponding to positive ΔpH) and the ΔpH becomes larger by decreasing the bulk pH as observed in HEWL. This suggests that pH dependence of the proton repulsion from the surface is a common phenomenon for the proteins with high pI. It is noted that the local structure around the probe and the relative position of charged residues in papain are different from those in HEWL. Therefore, the results in this study also suggest that not the local interaction around the probe but the net positive charge of the protein moiety (or at least some domain) determines the local pH at the surface of those proteins. Applying the quantitative analysis of the local pH performed here to the proteins with low pI, which is ongoing, will give us the comprehensive view about the local environment at the protein surface.
4. Conclusions In this study, pKa of FITC bound to HEWL was analyzed to elucidate the microenvironment at the HEWL surface. The result showed that the local pH at the HEWL surface is higher than the bulk pH due to the net positive charges and the consequent repulsion of protons from the HEWL surface. Furthermore, the difference between the local pH and the bulk pH becomes larger with decreasing the bulk pH. This pH-dependency can be attributed to the increase in the net positive charge of HEWL. The local pH affects the protonation state of amino-acid residues and vice versa, and the protonation/deprotonation of specific residues is a determinant for the effective enzymatic activity as 11
well as the efficient formation of protein-target complex [29]. Therefore, pH dependence of proton repulsion at the protein surface observed in this study provides the fundamental insight into various biological functions expressed by proteins.
Acknowledgements This work is partly supported by the Tenure-track program in Saitama University (SUTT project) and JSPS KAKENHI Grant Number 17K19097.
Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version.
References
[1] F. Fernandes, A. Coutinho, M. Prieto, L.M. Loura, Electrostatically driven lipid-protein interaction: Answers from FRET, Biochim. Biophys. Acta. 1848 (2015) 1837-1848. [2] F. Chiti, C.M. Dobson, Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem. 75 (2006) 333-366. [3] C.M. Dobson, Protein folding and misfolding, Nature 426 (2003) 884-890. [4] M. Stefani, C.M. Dobson, Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution, J. Mol. Med. 81 (2003) 678-699. [5] M. Bertsch, A.L. Mayburd, R.J. Kassner, The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue, Anal. Biochem. 313 (2003) 187-195. [6] B. Borucki, T. Lamparter, A polarity probe for monitoring light-induced structural changes at the rntrance of the chromophore pocket in a bacterial phytochrome, J. Biol. Chem. 284 (2009) 26005-26016. 12
[7] M.R. Eftink, J.L. Zajicek, C.A. Ghiron, A hydrophobic quencher of protein fluorescence: 2,2,2-trichloroethanol, Biochim. Biophys. Acta. 491 (1977) 473-481. [8] D. Toptygin, R.S. Savtchenko, N.D. Meadow, L. Brand, Homogeneous spectrally- and time-resolved fluorescence emission from single-tryptophan mutants of IIA(Glc) protein, J. Phys. Chem. B 105 (2001) 2043-2055. [9] D. Toptygin, T.B. Woolf, L. Brand, Picosecond protein dynamics: The origin of the time-dependent spectral shift in the fluorescence of the single trp in the protein GB1, J. Phys. Chem. B 114 (2010) 11323-11337. [10] S.K. Pal, J. Peon, B. Bagchi, A.H. Zewail, Biological water: Femtosecond dynamics of macromolecular hydration, J. Phys. Chem. B 106 (2002) 12376-12395. [11] S.K. Pal, J. Peon, A.H. Zewail, Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution, Proc. Natl. Acad. Sci. USA 99 (2002) 1763-1768. [12] D. Laage, T. Elsaesser, J.T. Hynes, Perspective: Structure and ultrafast dynamics of biomolecular hydration shells, Struct. Dynam. 4 (2017) 044018. [13] U. Alexiev, T. Marti, M.P. Heyn, H.G. Khorana, P. Scherrer, Surface-charge of bacteriorhodopsin detected with covalently bound pH indicators at selected extracellular and cytoplasmic sites, Biochemistry 33 (1994) 298-306. [14] R. Jonas, Y. Koutalos, T.G. Ebrey, Purple membrane - Surface-charge density and the multiple effect of pH and cations, Photochem. Photobiol. 52 (1990) 1163-1177. [15] E. Nishimoto, S. Yamashita, A.G. Szabo, T. Imoto, Internal motion of lysozyme studied by time-resolved fluorescence depolarization of tryptophan residues, Biochemistry 37 (1998) 5599-5607. [16] D. Takahashi, E. Nishimoto, T. Murase, S. Yamashita, Protein-protein interaction on lysozyme crystallization revealed by rotational diffusion analysis, Biophys. J. 94 (2008) 4484-4492. [17] B. van den Berg, R.J. Ellis, C.M. Dobson, Effects of macromolecular crowding on protein 13
folding and aggregation, Embo. J. 18 (1999) 6927-6933. [18] C. McAllister, M.A. Karymov, Y. Kawano, A.Y. Lushnikov, A. Mikheikin, V.N. Uversky, Y.L. Lyubchenko, Protein interactions and misfolding analyzed by AFM force spectroscopy, J. Mol. Biol. 354 (2005) 1028-1042. [19] K.H. Pearce, M. Hof, B.R. Lentz, N.L. Thompson, Comparison of the membrane-binding kinetics of bovine prothrombin and its fragment-1, J. Biol. Chem. 268 (1993) 22984-22991. [20] Z.M. Tamura, T; Maeda, M; Thuji, A, Spectrophotometric estimation of pKa values, absorption spectra and strucutral formulas of molecular species in aqueous solutions of fluorescein and sulfonefluorescein, Bunseki Kagaku 43 (1994) 339-346. [21] R. Sjoback, J. Nygren, M. Kubista, Absorption and fluorescence properties of fluorescein, spectrochim. Acta. A 51 (1995) L7-L21. [22] S. Yamaguchi, K. Bhattacharyya, T. Tahara, Acid-base equilibrium at an aqueous interface: pH spectrometry by heterodyne-detected electronic sum frequency generation, J. Phys. Chem. C 115 (2011) 4168-4173. [23] A. Kundu, S. Yamaguchi, T. Tahara, Evaluation of pH at charged lipid/water interfaces by heterodyne-detected electronic sum frequency generation, J. Phys. Chem. Lett. 5 (2014) 762-766. [24] L.R. Wetter, H.F. Deutsch, Immunological studies of egg white proteins. IV. Immunochemical and physical studies of lysozyme, J. Biol. Chem. 192 (1953) 237-242. [25] M.S. Fernandez, P. Fromherz, Lipoid pH indicators as probes of electrical potential and polarity in micelles, J. Phys. Chem. 81 (1977) 1755-1761. [26] G. Akerlof, Dielectric constants of some organic solvent-water mixtures at various temperatures, J. Am. Chem. Soc. 54 (1932) 4125-4139. [27] H. Webb, B.M. Tynan-Connolly, G.M. Lee, D. Farrell, F. O'Meara, C.R. Sondergaard, K. Teilum, C. Hewage, L.P. McIntosh, J.E. Nielsen, Remeasuring HEWL pK(a) values by NMR spectroscopy: methods, analysis, accuracy, and implications for theoretical pK(a) calculations, Proteins 79 (2011) 14
685-702. [28] L.A. Sluyterman, M.J. de Graaf, The effect of salts upon the pH dependence of the activity of papain and succinyl-papain, Biochim. Biophys. Acta. 258 (1972) 554-561. [29] T.K. Harris, G.J. Turner, Structural basis of perturbed pKa values of catalytic groups in enzyme active sites, IUBMB Life 53 (2002) 85-98.
15
samples
pKa1
4.15 FITC (7 vol% acetone) HEWL papain
3.79 3.82
*
*
ΔpH1
pKa2
ΔpH2
-
6.37
-
-
0.36 0.33
6.31 6.24
0.06 0.13
11.4 9.55
*
ΔpHi is calculated based on Eq. (5) using pKai
16
pI
Graphical abstract
17
Highlights Local pH at the surface of hen-egg-white lysozyme (HEWL) was examined. The local pH is higher than the bulk pH due to the net positive charge of HEWL. The pH difference becomes larger with decreasing the bulk pH.
18
S0009-2614(18)30034-4 https://doi.org/10.1016/j.cplett.2018.01.026 CPLETT 35378
To appear in:
Chemical Physics Letters
Received Date: Accepted Date:
19 December 2017 10 January 2018
Please cite this article as: T. Otosu, K. Kobayashi, S. Yamaguchi, Local pH at the surface of hen egg white lysozyme, Chemical Physics Letters (2018), doi: https://doi.org/10.1016/j.cplett.2018.01.026
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Local pH at the surface of hen egg white lysozyme
Takuhiro Otosu, Kaito Kobayashi, and Shoichi Yamaguchi* Department of Applied Chemistry, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura, Saitama 338-8570, Japan *[email protected]
Keywords: lysozyme, fluorescein, pH at the protein surface
1
Abstract The microenvironment at the surface of hen-egg-white lysozyme (HEWL) was examined by analyzing the change in pKa of fluorescein isothiocyanate (FITC) upon binding to the N-terminus of HEWL. The result showed that the local pH at the HEWL surface is higher than the bulk pH. Furthermore, the data showed that the difference between the local and bulk pH becomes larger with decreasing pH, suggesting HEWL repels more protons at lower pH. Because the local pH affects the protonation states of functional amino-acids at the protein surface, the results provide the fundamental insight into the microenvironment at the protein surface.
Highlights Local pH at the surface of hen-egg-white lysozyme (HEWL) was examined. The local pH is higher than the bulk pH due to the net positive charge of HEWL. The pH difference becomes larger with decreasing the bulk pH.
2
1. Introduction Various kinds of biological functions (e.g., signal transduction, replication and duplication of DNA, and respiration) achieved or mediated by proteins are initiated with the interaction between the protein surface and its target molecules. Attractive forces between these molecules due to electrostatic and/or hydrophobic interactions promote the effective formation of the protein-target complex [1]. On the other hand, strong attractive interaction causes the aggregate formation which leads to fatal diseases such as Alzheimer’s disease and Parkinson disease [2-4]. Therefore, various spectroscopic analyses have been attempted to elucidate the chemical properties of the microenvironment at the protein surface for understanding the sophisticated biological functions expressed by proteins. For example, steady-state absorption as well as fluorescence spectroscopies of tryptophan residue or some extrinsic probes have revealed the local polarities at the protein surface [5-7]. In addition to the static properties at the protein surface, time-resolved fluorescence spectral shift of tryptophan, which reflects the reorganization of water molecules surrounding the probe, has shown the dynamic nature of the microenvironment at the protein surface [8-12]. Vast of such studies have revealed that the dynamics of water at the protein surface is much slower than that of bulk water, which is often described as “biological” water [10,11]. Because biological water is expected to be excluded from the protein surface during the formation of protein-target complex, the reorganization dynamics of those water molecules has the fundamental insight into the kinetics of protein-target complex formation. Therefore, previous spectroscopic studies have been focused on the static and dynamic properties of the microenvironment at the protein surface. However, the electrostatic property of the protein surface, that is, local pH at the surface has not been extensively studied
except
for
bacteriorhodopsin
[13,14].
Because
the
local
pH
determines
the
protonation/deprotonation states of amino acids and target molecules located at the protein surface, estimation of this quantity is crucial to understand the various biological functions of proteins. In this letter, we analyzed the local pH at the surface of hen-egg-white lysozyme (HEWL) 3
Fig.
1.
X-ray
crystallographic
structure
of
hen-egg-white lysozyme (PDB ID: 3RZ4). Lysine residue
at
the
N-terminus,
where
fluorescein
isothiocyanate is bound, is shown by a stick.
through the change in the negative logarithm of the acid dissociation constant, pKa, of fluorescein isothiocyanate (FITC) upon covalent binding to the N-terminus of HEWL. HEWL is a small water-soluble protein which has been used as a model protein for protein folding, crystallization, and so on (Fig. 1) [15-18]. One remarkable feature of HEWL is its conformational stability against pH [18], which makes HEWL a suitable protein for analyzing the local pH at the surface of a folded protein via pH dependence of the absorption spectra of FITC bound to HEWL (hereafter referred to as FITC-Lz).
2. Materials and Methods Hen-egg-white lysozyme (HEWL) was purchased from Sigma-Aldrich, papain was purchased from Wako, and fluorescein isothiocyanate (FITC) was purchased from Dojindo. All reagents used in this study were of analytical grade and used without purification. To label HEWL with FITC, lyophilized powder of HEWL was dissolved into 20 mM sodium phosphate buffer (pH 7.5) containing 100 mM NaCl. The HEWL solution (~100 M) was then mixed with FITC solution (~50 4
mM) in dimethyl sulfoxide with a molar ratio of 1:2 and stirred for 2 h at room temperature. In this pH condition, FITC is expected to be selectively bound to the N-terminal -amine of HEWL [16]. To remove the unreacted FITC, the mixed solution was applied to PD-10 desalting column (GE Healthcare Life Sciences) that was equilibrated with 100 mM NaCl in water in advance. The labeling efficiency of FITC was estimated from the absorption spectrum of the sample and the extinction coefficient of HEWL (37800 M-1 cm-1 at 280 nm) and FITC (64800 M-1 cm-1 at 494 nm and 20400 M-1 cm-1 at 280 nm), and it was ~0.3 [16,19]. The FITC labeling of papain was performed with the same procedure as HEWL, and the labeling efficiency was ~0.4. For the absorption measurements, FITC-labeled proteins of ~20 M were diluted four times by using the following buffers depending on pH: 100 mM sodium phosphate buffer for pH 9.0–5.5, 100 mM sodium acetate buffer for pH 5.0–3.0, both of which contain 100 mM NaCl. Absorption spectra of those samples were then measured at 350–550 nm with the increment of 0.5 nm by using a UV-vis spectrophotometer (U-2900, HITACHI). Because the sample was limited and the protein might adsorb on the glass electrode of a pH meter (KR5EW, AS ONE), it was difficult to directly measure the bulk pH of the sample. Thus, the sample solution in the absence of labeled proteins was prepared by mixing 100 mM NaCl in water with the dilution buffer at the same mixing ratio as the sample and was used for the pH measurement to estimate the bulk pH of the sample. For the measurements of FITC in 7 vol% acetone, 20M FITC of 500 L in water containing 128 mM NaCl was mixed with the dilution buffer of 1360 L mentioned above and acetone of 140L. The bulk pH of the sample was also independently measured by preparing the same solution without FITC.
3. Results and Discussion Figure 2(a) shows the absorption spectra of FITC-Lz at various bulk pHs. At pH ~9.0, the spectrum shows the absorption maximum at ~490 nm with a smaller shoulder at ~455 nm. When pH 5
Fig. 2. (a) Absorption spectra of fluorescein isothiocyanate on the N-terminus of hen-egg-white lysozyme at various pHs. The bulk pH of each spectrum is shown in the figure. The data were fitted with Eqs. (1)-(4) and the reconstructed spectra are shown in (b).
is changed to pH 5.0, the band at ~490 nm decreases and is blue shifted to ~480 nm. Furthermore, a new peak at ~450 nm emerges. The absorbance at ~480 nm and ~450 nm decreases by further decreasing pH to ~3.0, and the latter peak is blue shifted to ~440 nm. Therefore, pH dependence of the absorption spectra of FITC-Lz suggests that three species with different absorption spectra are involved in the spectrum as reported for free fluorescein in this pH range [20,21]. To determine the pKa value of FITC-Lz, pH dependence of the absorption spectra shown in Fig. 2(a) was analyzed. At the pH range from 3 to 9, FITC can be regarded as a diprotic acid in successive equilibria [20,21]: H2A ⇄ H+ + HA- ⇄ 2H+ + A2-.
- scheme 1
Therefore, the absorption spectrum of the sample (A(pH, λ)) can be described with the theoretical equation for three-state equilibria as follows:
ApH,
AH 2A ( ) 10 pHpKa1 AHA ( ) 10 2 pHpKa1 pKa2 AA 2- ( ) 1 10 pHpKa1 10 2 pHpKa1 pKa2
.
(1)
In Eq. (1), pKa1 and pKa2 are the pKas between H2A and HA- and between HA- and A2-, respectively, and AH 2 A ( ) , AHA- ( ) , and AA 2- ( ) are the absorption spectra of H2A, HA- and A2-, respectively. 6
The absorption spectrum of each species can be described with a linear combination of experimentally obtained spectra at arbitrary pHs as follows: AH 2A ( ) 1 A(pH1 , ) 2 A(pH2 , ) 3 A(pH3 , ) ,
(2)
AHA- ( ) 1 A(pH1 , ) 2 A(pH2 , ) 3 A(pH3 , ) ,
(3)
AA 2- ( ) 1 A(pH1 , ) 2 A(pH2 , ) 3 A(pH3 , ) .
(4)
In the actual analysis, pH1, pH2, and pH3 were set to 3.1, 6.1, and 8.9, respectively. All spectra obtained at different pHs were then globally fitted by using Eqs. (1)-(4), and the fitting parameters pKa1, pKa2, i, i, and i (i = 1-3) were determined. Figure 2(b) shows the reconstructed spectra of FITC-Lz that were obtained by the global fitting of the experimental data with Eqs. (1)-(4). (The absorption spectra of H2A, HA- and A2- are shown in Fig. S1.) Agreement between the experimental and reconstructed spectra confirms that the three-state model (scheme 1) is adequate to reproduce the essential features of the spectral change such as the peak shift and the existence of the isosbestic point at ~460 nm. Based on the fitting, pKa1 and pKa2 were determined to be 3.79 and 6.31, respectively. It is important to note that the obtained pKa is the “apparent” one because the data shown in Fig. 2(a) were analyzed by using the bulk pH that is not necessarily the same as the pH that the probe experiences. Actually, recent sum-frequency generation studies of the charged surfaces have shown that the local pH at the charged surface is different from the bulk pH due to the electrostatic repulsion/attraction of protons from/to the surface [22,23]. Indeed, the isoelectric point (pI, the pH where the net charge becomes zero) of HEWL is 11.35 and HEWL is positively charged at the pH range examined in this study [24]. Thus, the local pH at the HEWL surface is anticipated to be higher than the bulk pH. While the apparent pKa was determined with respect to the bulk pH, the “intrinsic” pKa of the probe at the protein surface is defined with respect to the local pH. The intrinsic pKa is thus described using the apparent one as follows, taking account of the difference (ΔpH) between the 7
local pH and the bulk pH:
Fig. 3. (a) Absorption spectra of fluorescein isothiocyanate (FITC) in different water-acetone mixture at pH 9.0. For comparison, the spectrum of FITC on hen-egg-white lysozyme (HEWL) measured at pH 8.9 is also shown (black solid line). Data are normalized to match the absorption maximum. (b) Relationship among the absorption peak wavelength of FITC, the dielectric constant of the solvent, and the volume percentage of acetone in water-acetone mixture. Data (open circle) were obtained from (a), and fitted with a linear function (red broken line). Peak wavelength of FITC on HEWL at pH 8.9 is also plotted on the fitting data (black rectangle).
intrinsic pKa = apparent pKa + local pH – bulk pH = apparent pKa + ΔpH.
(5)
Therefore, the evaluation of intrinsic pKa is needed to get the information of the local pH. Because pKa is given by the standard reaction Gibbs energy from the acid to the base, intrinsic pKa is directly related to the local effective polarity (or dielectric constant) of medium surrounding the probe [25]. Thus, the effective dielectric constant at the probe position was first examined by measuring the solvatochromic shift of the FITC absorption spectrum upon binding to the HEWL surface. Figure 3(a) shows the absorption spectra of free FITC in solution containing different vol% of acetone. The samples were prepared by mixing FITC in sodium phosphate buffer at pH 9.0 with acetone. At this pH, A2- is a dominant species in FITC. The spectrum of A2- is shifted to longer wavelength by increasing the mixing ratio of acetone (decreasing the dielectric constant of the solvent), which indicates negative solvatochromism. Therefore, this result suggests that the peak wavelength of A2- is a good indicator to estimate the effective dielectric constant at the protein 8
Fig.
4.
Absorption
spectra
of
fluorescein
isothiocyanate in 7 vol% acetone at various pHs. The bulk pH of each spectrum is shown in the figure. The data were fitted with Eqs. (1)-(4) and the reconstructed spectra are shown by broken lines.
surface. Figure 3(b) shows the relationship between the dielectric constant of the solvent and the peak wavelengths of FITC. The data were fitted with a linear function and the result is also shown in the figure. The dielectric constants of water-acetone mixture were obtained from literature [26]. Based on the fitting and the peak wavelength of FITC-Lz at pH ~9.0, it was found that the effective dielectric constant at the HEWL surface is equivalent to that in 7 vol% acetone in water. It is worth noting that the conformation of HEWL is robust and insensitive to pH from 3.0 to 9.0 [18]. Therefore, it is reasonable to assume that the effective dielectric constant at the HEWL surface is independent of pH. To determine the intrinsic pKa of FITC-Lz, the absorption spectra of free FITC in 7 vol% acetone at various pHs were measured. The results are shown in Fig. 4. As expected, the absorption maximum at pH 9.0 was the same as that in FITC-Lz at the same pH. Based on the fitting of the data shown in Fig. 4, pKa1 and pKa2 were determined to be 4.15 and 6.37, respectively, which correspond to the intrinsic pKas of FITC bound to HEWL. Using Eq. (5), ΔpH (= intrinsic pKa – apparent pKa) was then calculated to be 0.36 at around the bulk pH ~4 and 0.06 at around the bulk pH ~6. Positive ΔpH indicates that the local pH is higher than the bulk pH, that is, a proton is repelled from the HEWL surface. It is reasonable because the pI of HEWL is ~11.4 and thus HEWL possesses a net positive charge at the pH region measured in this study. Interestingly, the result also showed that ΔpH 9
Fig. 5. (a) Absorption spectra of fluorescein isothiocyanate on the N-terminus of papain at various pHs. The bulk pH of each spectrum is shown in the figure. The data were fitted with Eqs. (1)-(4) and the reconstructed spectra are shown in (b).
becomes larger by decreasing the bulk pH from ~6 to ~4. Because positive ΔpH arises from the proton repulsion from the HEWL surface, this pH dependency can be ascribed to the increase in the net positive charge of HEWL upon decreasing the bulk pH. In other words, the result suggests that some ionizable residues in HEWL are protonated (or neutralized from the base form) by changing the bulk pH from 6 to 4. Actually, Webb et al. analyzed the pKa of ionizable amino-acid residues in HEWL and showed that several amino acids such as His15 have pKa in between pH 6 and 4 [27]. Therefore, the change in the protonation states of such amino acids might be reflected on the pH dependence of the proton repulsion from the HEWL surface. It is tempting to think whether this observation is specific to HEWL or rather general for the protein with a high pI. Actually, the N-terminal amino acid of HEWL is a lysine residue so that the specific interaction between FITC and the amino group in the lysine residue might contribute to the ΔpH observed here. In this regard, we also analyzed the local pH at the papain surface. Papain is also a water-soluble small protein which has a pI of 9.55 and possesses an isoleucine residue on its N-terminus [28]. The absorption spectra of FITC-papain and these fitting curves with Eqs. (1)-(4) are shown in Fig. 5. The peak wavelength at pH ~9.0 was the same as that of FITC-Lz, suggesting that 10
Table 1 Fitting results and the isoelectric points (pI) of proteins
the effective dielectric constant at the probe position was equivalent to that of FITC-Lz (corresponding to 7 vol% acetone). Fitting results are summarized in Table 1. The results showed that the apparent pKas of papain are lower than the intrinsic ones (corresponding to positive ΔpH) and the ΔpH becomes larger by decreasing the bulk pH as observed in HEWL. This suggests that pH dependence of the proton repulsion from the surface is a common phenomenon for the proteins with high pI. It is noted that the local structure around the probe and the relative position of charged residues in papain are different from those in HEWL. Therefore, the results in this study also suggest that not the local interaction around the probe but the net positive charge of the protein moiety (or at least some domain) determines the local pH at the surface of those proteins. Applying the quantitative analysis of the local pH performed here to the proteins with low pI, which is ongoing, will give us the comprehensive view about the local environment at the protein surface.
4. Conclusions In this study, pKa of FITC bound to HEWL was analyzed to elucidate the microenvironment at the HEWL surface. The result showed that the local pH at the HEWL surface is higher than the bulk pH due to the net positive charges and the consequent repulsion of protons from the HEWL surface. Furthermore, the difference between the local pH and the bulk pH becomes larger with decreasing the bulk pH. This pH-dependency can be attributed to the increase in the net positive charge of HEWL. The local pH affects the protonation state of amino-acid residues and vice versa, and the protonation/deprotonation of specific residues is a determinant for the effective enzymatic activity as 11
well as the efficient formation of protein-target complex [29]. Therefore, pH dependence of proton repulsion at the protein surface observed in this study provides the fundamental insight into various biological functions expressed by proteins.
Acknowledgements This work is partly supported by the Tenure-track program in Saitama University (SUTT project) and JSPS KAKENHI Grant Number 17K19097.
Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version.
References
[1] F. Fernandes, A. Coutinho, M. Prieto, L.M. Loura, Electrostatically driven lipid-protein interaction: Answers from FRET, Biochim. Biophys. Acta. 1848 (2015) 1837-1848. [2] F. Chiti, C.M. Dobson, Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem. 75 (2006) 333-366. [3] C.M. Dobson, Protein folding and misfolding, Nature 426 (2003) 884-890. [4] M. Stefani, C.M. Dobson, Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution, J. Mol. Med. 81 (2003) 678-699. [5] M. Bertsch, A.L. Mayburd, R.J. Kassner, The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue, Anal. Biochem. 313 (2003) 187-195. [6] B. Borucki, T. Lamparter, A polarity probe for monitoring light-induced structural changes at the rntrance of the chromophore pocket in a bacterial phytochrome, J. Biol. Chem. 284 (2009) 26005-26016. 12
[7] M.R. Eftink, J.L. Zajicek, C.A. Ghiron, A hydrophobic quencher of protein fluorescence: 2,2,2-trichloroethanol, Biochim. Biophys. Acta. 491 (1977) 473-481. [8] D. Toptygin, R.S. Savtchenko, N.D. Meadow, L. Brand, Homogeneous spectrally- and time-resolved fluorescence emission from single-tryptophan mutants of IIA(Glc) protein, J. Phys. Chem. B 105 (2001) 2043-2055. [9] D. Toptygin, T.B. Woolf, L. Brand, Picosecond protein dynamics: The origin of the time-dependent spectral shift in the fluorescence of the single trp in the protein GB1, J. Phys. Chem. B 114 (2010) 11323-11337. [10] S.K. Pal, J. Peon, B. Bagchi, A.H. Zewail, Biological water: Femtosecond dynamics of macromolecular hydration, J. Phys. Chem. B 106 (2002) 12376-12395. [11] S.K. Pal, J. Peon, A.H. Zewail, Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution, Proc. Natl. Acad. Sci. USA 99 (2002) 1763-1768. [12] D. Laage, T. Elsaesser, J.T. Hynes, Perspective: Structure and ultrafast dynamics of biomolecular hydration shells, Struct. Dynam. 4 (2017) 044018. [13] U. Alexiev, T. Marti, M.P. Heyn, H.G. Khorana, P. Scherrer, Surface-charge of bacteriorhodopsin detected with covalently bound pH indicators at selected extracellular and cytoplasmic sites, Biochemistry 33 (1994) 298-306. [14] R. Jonas, Y. Koutalos, T.G. Ebrey, Purple membrane - Surface-charge density and the multiple effect of pH and cations, Photochem. Photobiol. 52 (1990) 1163-1177. [15] E. Nishimoto, S. Yamashita, A.G. Szabo, T. Imoto, Internal motion of lysozyme studied by time-resolved fluorescence depolarization of tryptophan residues, Biochemistry 37 (1998) 5599-5607. [16] D. Takahashi, E. Nishimoto, T. Murase, S. Yamashita, Protein-protein interaction on lysozyme crystallization revealed by rotational diffusion analysis, Biophys. J. 94 (2008) 4484-4492. [17] B. van den Berg, R.J. Ellis, C.M. Dobson, Effects of macromolecular crowding on protein 13
folding and aggregation, Embo. J. 18 (1999) 6927-6933. [18] C. McAllister, M.A. Karymov, Y. Kawano, A.Y. Lushnikov, A. Mikheikin, V.N. Uversky, Y.L. Lyubchenko, Protein interactions and misfolding analyzed by AFM force spectroscopy, J. Mol. Biol. 354 (2005) 1028-1042. [19] K.H. Pearce, M. Hof, B.R. Lentz, N.L. Thompson, Comparison of the membrane-binding kinetics of bovine prothrombin and its fragment-1, J. Biol. Chem. 268 (1993) 22984-22991. [20] Z.M. Tamura, T; Maeda, M; Thuji, A, Spectrophotometric estimation of pKa values, absorption spectra and strucutral formulas of molecular species in aqueous solutions of fluorescein and sulfonefluorescein, Bunseki Kagaku 43 (1994) 339-346. [21] R. Sjoback, J. Nygren, M. Kubista, Absorption and fluorescence properties of fluorescein, spectrochim. Acta. A 51 (1995) L7-L21. [22] S. Yamaguchi, K. Bhattacharyya, T. Tahara, Acid-base equilibrium at an aqueous interface: pH spectrometry by heterodyne-detected electronic sum frequency generation, J. Phys. Chem. C 115 (2011) 4168-4173. [23] A. Kundu, S. Yamaguchi, T. Tahara, Evaluation of pH at charged lipid/water interfaces by heterodyne-detected electronic sum frequency generation, J. Phys. Chem. Lett. 5 (2014) 762-766. [24] L.R. Wetter, H.F. Deutsch, Immunological studies of egg white proteins. IV. Immunochemical and physical studies of lysozyme, J. Biol. Chem. 192 (1953) 237-242. [25] M.S. Fernandez, P. Fromherz, Lipoid pH indicators as probes of electrical potential and polarity in micelles, J. Phys. Chem. 81 (1977) 1755-1761. [26] G. Akerlof, Dielectric constants of some organic solvent-water mixtures at various temperatures, J. Am. Chem. Soc. 54 (1932) 4125-4139. [27] H. Webb, B.M. Tynan-Connolly, G.M. Lee, D. Farrell, F. O'Meara, C.R. Sondergaard, K. Teilum, C. Hewage, L.P. McIntosh, J.E. Nielsen, Remeasuring HEWL pK(a) values by NMR spectroscopy: methods, analysis, accuracy, and implications for theoretical pK(a) calculations, Proteins 79 (2011) 14
685-702. [28] L.A. Sluyterman, M.J. de Graaf, The effect of salts upon the pH dependence of the activity of papain and succinyl-papain, Biochim. Biophys. Acta. 258 (1972) 554-561. [29] T.K. Harris, G.J. Turner, Structural basis of perturbed pKa values of catalytic groups in enzyme active sites, IUBMB Life 53 (2002) 85-98.
15
samples
pKa1
4.15 FITC (7 vol% acetone) HEWL papain
3.79 3.82
*
*
ΔpH1
pKa2
ΔpH2
-
6.37
-
-
0.36 0.33
6.31 6.24
0.06 0.13
11.4 9.55
*
ΔpHi is calculated based on Eq. (5) using pKai
16
pI
Graphical abstract
17
Highlights Local pH at the surface of hen-egg-white lysozyme (HEWL) was examined. The local pH is higher than the bulk pH due to the net positive charge of HEWL. The pH difference becomes larger with decreasing the bulk pH.
18