Effect of Charge Regulation and Ion–Dipole Interactions on the

Feb 28, 2014 - Department of Materials Science and Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden. § Centre ... We investiga...
0 downloads 17 Views 861KB Size
Article pubs.acs.org/Langmuir

Effect of Charge Regulation and Ion−Dipole Interactions on the Selectivity of Protein−Nanoparticle Binding Fernando Luís Barroso da Silva,*,† Mathias Boström,‡,§ and Clas Persson‡,§,# †

Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences at Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP 14040-903, Brazil ‡ Department of Materials Science and Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden § Centre for Materials Science and Nanotechnology, University of Oslo, P.O. Box 1048 Blindern, NO-0316 Oslo, Norway # Department of Physics, University of Oslo, P.O. Box 1048 Blindern, NO-0316 Oslo, Norway ABSTRACT: We investigate the role of different mesoscopic interactions (Coulomb, charge regulation, and ion-dipole “surface patch” effects) on the binding of bovine serum albumin (BSA) and β-lactoglobulin (BLG) to a cationic gold nanoparticle (TTMA+). The results demonstrate that the charge-regulation mechanism plays a vital role for selectivity of protein−nanoparticle complexation at low salt concentration. At slightly higher ionic strengths, charge-dipole effects are the dominating driving force. Thus, very small variations in salt concentration strongly influence the origin of complexation.



the charge regulation mechanism9,14,21,22 and (b) the “charge patch” mechanism due the protein anisotropy.5,21 These two mechanisms can, under specific conditions, give rise to complexation also when the protein (P) and nanoparticle (NP) are both positively (or negatively) charged. This phenomenon is known in the literature as “binding (or complexation) on the wrong side of the isoelectric point”.1,5,23 These interactions occur between materials of an intermediate length scale, i.e., proteins and nanoparticles. Therefore, they are usually referred to as “mesoscopic interactions”.24,25 Both are frequently claimed to be the key mechanism responsible for the complexation phenomena in the literature9,14,21,22,26,27 and were numerically scrutinized here. This is the main reason to concentrate our present study on pure Coulombic interactions, and not include at this stage ion specificity into the models. As will be shown here, we find that Dubin and co-workers4,5 were correct in assuming that an important driving force behind the P−NP complexation is the charge-dipole interaction at moderate and high ionic strengths. However, at extremely low salt concentration, we find that the charge regulation mechanism often dominates the interactions. At low salt concentrations, both contributions can be equally important. In general, the two mechanisms together enhance attraction. Consequently, this study defines when each of these physical mechanisms plays a key role driving the macromolecular complexation.

INTRODUCTION Protein adsorption, precipitation, and separation are important in food science, biosensors, biotechnology, and biomedical applications.1−5 Extensive work has focused on the role of solution pH, salt type, and concentration on protein−protein and protein−surface interactions.6−9 These are key variables for design and optimization in protein separation and purification.7,8 They also influence macromolecular complexation between biological molecules (e.g., proteins, DNA, RNA, or vaccine) and nanoparticles. The importance of protein− nanoparticle (P−NP) interactions in bionanotechnology and medicine10−13 has led to an intense search for experimental and theoretical tools that enable fast and easy to use estimates of the protein charge and the complexation phenomena. We investigate here the role of Coulomb interactions, charge regulation,14,15 and charge-dipole effects (due to anisotropic surface patches) on the binding of bovine serum albumin (BSA) and β-lactoglobulin (BLG) to a cationic gold nanoparticle coupled to 3,6,9,12-tetraoxatricosan-1-aminium, 23mercapto-N,N,N-trimethyl. Having the tetra-ethylene glycoltrimethyl-amine (TTMA) attached to this NP, the term “TTMA” is also used to refer to this NP.5 Here, we will use “TTMA+” to refer to this cationic NP. The ion-specific dependence of BSA protein charge on salt solution has recently been reinvestigated by potentiometric titrations and electrophoretic light scattering measurements.16 It has been known for a long time that protein charge and precipitation can be modified by changes in salt concentration or pH, and that these effects follow ion specific Hofmeister sequences.16−20 We focus here on the salt concentration and pH effects. Currently, there are two theoretical frameworks to explain their effects on the macromolecular complexation phenomena: (a) © 2014 American Chemical Society

Received: November 6, 2013 Revised: February 25, 2014 Published: February 28, 2014 4078

dx.doi.org/10.1021/la500027f | Langmuir 2014, 30, 4078−4083

Langmuir

Article

Figure 1. The simulated charge number (Z), the protein capacitance (C), and the dipole moment number (μ) as a function of pH for BSA at different salt concentrations. the literature.9,15,22 If the NP has chemical groups that can also titrate, then the attractive term of eq 1 will be enhanced, and even larger effects will be observed. We assume here that any of such groups have pKs smaller than the studied pHs. In the presence of salt, the screening effect is taken into account, and A(R) can be estimated as follows:9

We now outline briefly the theory and give some illustrative numerical results that demonstrate the relative roles played by each of these mechanisms on the selectivity of P−NP binding, as mentioned above. Although our discussion will be centered on the BSA-TTMA+ and BLG-TTMA+ systems,5 our findings can be generalized to any protein-charged macroion system in an electrolyte solution.



βA(R ) ≈

THEORETICAL METHODS

2⎧ ⎧ exp[− κ(R − d)] ⎫2 ⎫2 2 ⟨μ⟩0 exp[ − κ(R − d)] ⎨ ⎬ − lB2Z NP ⎨ ⎬ (1 + κd) (1 + κd) ⎩ ⎭ ⎭ 6R4 ⎩

Proteins are ionizable objects that can titrate in electrolyte solutions. This implies that charge can fluctuate particularly at pH = pI, which gives an additional pure electrostatic attraction when they are interacting with another charged object (e.g., TTMA+) that results in the macromolecular association. Since this interaction occurs between materials of an intermediate length scale, i.e., molecules, nanoparticles, they are usually referred to as “mesoscopic interactions”.24,25 According to Kirkwood and Shumaker (KS),28 the electrostatic free energy [A(R)] for the P−NP complexation may be written (in the limit of no salt) in a perturbation theory framework (details are given elsewhere9,15,29) as a function of their center-to-center separation distance (R) as follows:

βA(R ) ≈

⎛ C lBZ NP⟨Z⟩0 ⟨μ⟩20 ⎞ 2 ⎟ ⎜ 2 + − lB2Z NP R 6R4 ⎠ ⎝ 2R

2 lBZ NP⟨Z⟩0 ⎧ exp[− κ(R − d)] ⎫ lB2Z NP C ⎨ ⎬− R (1 + κd) ⎩ ⎭ 2R2

(1 + κR )2

(2)

where κ is the Debye−Hückel screening parameter given by 0.329(cs)1/2 [Å−1], cs is the 1:1 salt concentration (in M), RP is the protein radius, RNP is the NP radius, and d = (RP + RNP)/2. In the first, second, and third terms of eq 2, we refer to, respectively, as ACou (ordinary Coulombic interaction), Areg (charge regulation), and Adip (ion-dipole). The ratio ϕ = Areg(R)/Adip(R) will be used as a simple manner to quantify the different contributions assuming that both Areg(R) and Adip(R) were obtained at the same experimental conditions (pH, cs, etc.).21 In order to mimic TTMA+,5 we have used RNP = 50 Å and ZNP = 100. It is assumed that the gold core together with its TTMA molecules define a charged hard-sphere (macroion), and there is no Helmholtz plane of strongly associated anions at the gold particle surface. The protein radii were 50.7 and 42.4 Å for BSA and BLG (dimeric form), respectively. The εs was assumed to be equal to 78.7 at T = 298.15 K. The salt concentration was varied from 0 to 250 mM. All other parameters were obtained from protein titration simulations by Monte Carlo (MC) within the cell model framework (see ref 21 for details). We used the following Protein Data Bank (PDB)30 identities for their three-dimensional structures: 1BEB for BLG and 1AO6 for

(1)

where β = 1/kBT, lB = e /4πε0εskBT is the Bjerrum length, e is the elementary charge, ε0 is the vacuum permittivity, εs is aqueous solution dielectric constant, T is the temperature, ZNP is the NP average valence, ⟨Z⟩0 is the protein average valence, ⟨μ⟩0 = |∑iziri| the average dipole moment number, and finally, C is a measurement of the protein charge fluctuations due the acid−base equilibrium. This is an intrinsic protein property that has been called the protein charge capacitance in 2

4079

dx.doi.org/10.1021/la500027f | Langmuir 2014, 30, 4078−4083

Langmuir

Article

Figure 2. The simulated charge number (Z), the protein capacitance (C), and the dipole moment number (μ) as a function of pH for BLG (dimer) at different salt concentrations. BSA. All protein atoms present in these structures were used in the calculations without further modification. They were described by hard spheres of radius Ra = 2 Å in the MC runs. The protein is kept fixed at the center of the simulation cell. Its coordinates remain unchanged, neglecting structural conformation changes. Protein atomic charges are varied according to the pH and the acid−base equilibrium.31 The dissociation constants for the single ionizable amino acids were taked from ref 32. The simulation cell radius was 150 Å for all systems. Explicit free ions (salt particles and counterions) described by the restricted primitive model33 were added to the system in order to make the simulation cell electroneutral. Equilibration and production runs were carried out in a semigrand canonical ensemble with at least 108 steps using the standard Metropolis algorithm.34



experimental data for pH values close to the isoionic point was that the increase of salt concentrations results in a higher experimental BSA surface charge at a certain pH. On the contrary, a lower BSA zeta potential is obtained as salt concentration is increased.16 Therefore, the surface charge of proteins depend on the way it is measured and on the way different ions interact with the protein charge groups. Both proteins show peaks for their C values at almost the same pHs. Nevertheless, BSA has higher capacitances. As a consequence, the charge regulation contribution has a large effect for BSA at these corresponding pH values. BSA also presents the higher dipole moments numbers. At pH 7.5, μBSA is ca. 350. This confirms the ability of this protein to easily form macromolecular complexes. The salt concentration has an effect on the dipole moments, increasing them particularly at very acid and basic regions. Therefore, it is expected to see a more pronounced contribution from the ion-dipole interaction at higher ionic strenghts. Assuming that the criterion for the complexation is A(R) < −1kBT, we can define pHC as the smallest pH when this happens at each salt concentration. Applying the simulated charge numbers, the protein capacitances and the dipole moment numbers obtained at different ionic strengths and pHs in eq 2 results in A(R) for each of such experimental conditions. From these data, pHC can be determined. Once pHC is known, the protein charge number at pH = pHC, ZC, and all the other quantities can also be determined. The plot of pHC and ZC at different salt concentrations are for separation distances of R = 100.7 and 92.4 Å for BSA and BLG, respectively (see Figure 3).

RESULTS FROM SIMULATIONS AND THEORY

The titration, capacitance, and dipole moment number plots for BSA and BLG obtained by the MC simulations are shown in Figures 1 and 2, respectively. The corresponding pIs for them are 5.5 and 4.5 at 5 mM. The experimental data can be rather scattered, as shown before.16 Different pI values have been reported for BLG (e.g., 4.6,35 4.8,36 5.1−5.516) and BSA (e.g., 4.7−5.616). Increasing the salt concentration to 250 mM shifts down these values to 5.4 and 4.3. BSA can be more highly charged than BLG. Even though some quantitative discrepancy between the simulated and experimental data could be expected due the model simplifications (e.g., the protein coordinates are kept unmodified at all pHs) and direct experimental uncertainties, the salt dependence is in agreement with experimental measurements.16 Particularly, the BSA protein charge from simulations follows the same general trends as observed in experiments.16 An interesting observation on 4080

dx.doi.org/10.1021/la500027f | Langmuir 2014, 30, 4078−4083

Langmuir

Article

Figure 4. The electrostatic free energy βA(R = d) and ϕ at different pH values for BLG. The salt concentration is 5 mM for the βA(R) data. ϕ is also given at 55 mM.

Figure 3. The electrostatic free energy βA(R = d) and ϕ at different pH values for BSA. The salt concentration is 5 mM for the βA(R) data. ϕ is also given at 55 mM.

At low salt concentration, we can confirm by the present semiquantitative analysis the observed experimental behavior of the complexation on the wrong side.5 Chen and coauthors5 have experimentally shown that both BSA and BLG can form complexes with TTMA+ at pH < pI. They have measured pHC and ZC for BSA, equal approximately to 4.2 and 6, respectively, at low salt concentration. For BLG, their outcomes were approximately 4.7 and 10 at the same experimental conditions. Despite the simplifications of our theoretical framework, the same trends are reproduced. For cs < 50 mM, both BSA and BLG are positively charged and form complexes with TTMA+. The charge regulation contribution is the main driven mechanism to overcome the repulsive ion−ion interaction at low salt concentration. ϕBSA and ϕBLG are equal to 7.2 (pH = 2.9) and 8.6 (pH = 2.6), respectively, in the limit of no salt. Conversely, these values drop down to 0.3 (pH = 4.3) and 0.7 (pH = 3.9) at 5 mM and essentially vanish at higher ionic strengths. At 5 mM and pH 4.3 (ZBSA = +11.0), although ACou(R) is equal to +11.1kT, A(R) = −1.4 kT, due the attractive contributions from the Areg(R) (= −2.8 kT) and the Adip(R) (= −9.7 kT) terms. The same is observed for BLG at pH 3.9 (ZBLG = +5.5), where the values for A(R), ACou(R), Areg(R), and Adip(R) are, respectively, −0.8, +7.0, −3.3, and −4.5 kT. From these plots, it can be seen that BLG tends to form complexes at smaller pH values in comparison to BSA (e.g., pHc,BLG = 3.9 and pHc,BSA = 4.3 at 5 mM, and pHc,BLG = 9.8 and pHc,BSA = 10.3 at 55 mM). For cs > 80 mM, BLG complexes due ordinary attractive ion−ion interactions. BSA is able to resist more the salt screening effect due its higher dipole and capacitance. These results are consistent with the study of Dubin and co-workers.4 Plots of βA(R = d) and ϕ at different pH values and low salt concentration for BSA and BLG are given in Figures 4 and 5, respectively. The partial contributions from the ordinary Coulombic interaction (ACou), the charge regulation (Areg), and the ion-dipole (Adip) are presented together with the total free energy Atot (=ACou + Areg + Adip). It can be observed that Atot is shifted to the left for both protein systems due the extra electrostatic attraction given by the Areg and Adip terms. For BSA, the difference between ACou = 0 and Atot = 0 corresponds to 1.3 pH units at cs = 5 mM. This means that the complexation can start at pH regimes where both macroions have net charges with identical sign. At pH = 4.3 and cs = 5 mM, the net charge

Figure 5. The smallest pH where complexation occurs (pHc) and the corresponding charge number of complexation (Zc) of BSA and BLG proteins with TTMA+.

number of BSA is +11. The corresponding pH displacement for BLG is 0.7. Quantifying the contributions from the charge regulation and ion-dipole terms by ϕ reveals that the ion-dipole term plays a key role for BSA in most of the studied pH values at the low salt concentration regime, due to the BSA high dipole moment. Conversely, the charge regulation term is the dominant mechanism for the BLG-TTMA+ complexation under the same experimental conditions. In some pH values, ϕ can reach above 8. An increase in the ionic strength has a considerable effect on the screening of the charge regulation term. As can be seen in the ϕ plots, at cs = 55 mM, the picture for both protein systems is essentialy dominated by the ion-dipole contribution. At higher salt concentration, this effect is even more pronounced (data not shown due the graphic scale). However, it should be noted that repeating the same analysis at the no salt limit regime would show that the charge regulation is the leader contribution even for BSA. The electrostatic selectivety can be tuned by changing the charge and size of the NP. For instance, if ZNano = +150, then the complexation at 5 mM starts already at pH 3.6 for BLG (ZBLG = +8.3, ϕBLG = 0.8) and at pH 3.9 for BSA (ZBSA = +16.5, 4081

dx.doi.org/10.1021/la500027f | Langmuir 2014, 30, 4078−4083

Langmuir

Article

ϕBSA = 0.4). A smaller NP can also reduce pHc. RNano = 30 Å brings pHc to 3.3 (ZBLG = +11.2, ϕBLG = 0.7) and 3.6 (ZBSA = +21.4, ϕBSA = 0.4) for BLG and BSA, respectively. The experimental work done by Chen and coauthors,5 whose main physical mechanisms were elucidated here, does not discuss ion dispersion interactions that might also play a role. Therefore, we propose that experiments of the kind performed by Medda et al.,16 combined with simulations that include ionspecific potentials between ions and discrete protein charge groups, could be used to study how the choice of different monovalent salt solutions influence complexation. It could be possible to optimize the conditions for protein−NP complexation, not only by salt concentration and pH, but also by changing the ionic species of the background salt. We plan to investigate this in the near future, following the work of Deniz and Parsons.20

(4) Xu, Y.; Mazzawi, M.; Chen, K.; Sun, L.; Dubin, P. L. Protein Purification by Polyelectrolyte Coacervation: Influence of Protein Charge Anisotropy on Selectivity. Biomacromolecules 2011, 12, 1512− 1522. (5) Chen, K.; Xu, Y.; Rana, S.; Miranda, O. R.; Dubin, P. L.; Rotello, V. M.; Sun, L.; Guo, X. Electrostatic Selectivity in ProteinNanoparticle Interactions. Biomacromolecules 2011, 12, 2552−2561. (6) Kunz, W.; Henle, J.; Ninham, B. W. Zur Lehre von der Wirkung der Salze (about the Science of the Effect of Salts): from Hofmeisterʼs Historical Papers. Curr. Opin. Colloid Interface Sci. 2004, 9, 19−37. (7) Chiew, Y. C.; Blanch, H. W.; Prausnitz, J. M. Molecular Thermodynamics for Salt-Induced Protein Precipitation. AIChE J. 1995, 41, 2150−2159. (8) Tardieu, A.; Bonnete, F.; Finet, D. S.; Vivares, D. Understanding Salt or PEG Induced Attractive Interactions to Crystallize Biological Macromolecules. Acta Crystallogr. D−Biol. Crystallogr. 2002, 58, 1549− 1553. (9) Jönsson, B.; Lund, M.; da Silva, F. L. B. Electrostatics in Macromolecular Solution. Food Colloids: Self-Assembly Mater. Sci., London, 2007; pp 129−154. (10) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14265−14270. (11) Parveen, S.; Misra, R.; Sahoo, S. K. Nanoparticles: a Boon to Drug Delivery, Therapeutics, Diagnostics and Imaging. Nanomed.: Nanotechnol., Biol. Med. 2012, 8, 147−166. (12) Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A. Biomolecular Coronas Provide the Biological Identity of Nanosized Materials. Nat. Nanotechnol. 2012, 7, 779−786. (13) Khan, S.; Gupta, A.; Nandi, C. K. Controlling the Fate of Protein Corona by Tuning Surface Properties of Nanoparticles. J. Phys. Chem. Lett. 2013, 4, 3747−3752. (14) Biesheuvel, P. M.; Stuart, M. A. C. Electrostatic Free Energy of Weakly Charged Macromolecules in Solution and Intermacromolecular Complexes Consisting of Oppositely Charged Polymers. Langmuir 2004, 20, 2785. (15) Lund, M.; Jönsson, B. On the Charge Regulation of Proteins. Biochemistry 2005, 44, 5722−5727. (16) Medda, L.; Barse, B.; Cugia, F.; Boström, M.; Parsons, D. F.; Ninham, B. W.; Monduzzi, M.; Salis, A. Hofmeister Challenges: Ion Binding and Charge of the BSA Protein as Explicit Examples. Langmuir 2012, 28, 16355−16363. (17) Ninham, B.; Yaminsky, V. Ion Binding and Ion Specificity: The Hofmeister Effect and Onsager and Lifshitz Theories. Langmuir 1997, 13, 2097. (18) Tavares, F. W.; Bratko, D.; Blanch, H. W.; Prausnitz, J. M. IonSpecific Effects in the Colloid-Colloid or Protein-Protein Potential of Mean Force: Role of Salt-Macroion van der Waals Interactions. J. Phys. Chem. B 2004, 108, 9228−9235. (19) Tanford, C.; Roxby, R. Interpretation of Protein Titration Curves. Application to Lysozyme. Biochemistry 1972, 11, 2192−2198. (20) Deniz, V.; Parsons, D. F. Effect of Nonelectrostatic Ion Interactions on Surface Forces Involving Ion Adsorption Equilibria. J. Phys. Chem. C 2013, 117, 16416−16428. (21) Da Silva, F. L. B.; Jö nsson, B. Polyelectrolyte-protein complexation driven by charge regulation. Soft Matter 2009, 5, 2862−2868. (22) Lund, M.; Jönsson, B. Charge Regulation in Biomolecular Solution. Q. Rev. Biophys. 2013, 46, 265−281. (23) Japrung, D.; Dogan, J.; Freedman, K. J.; Nadzeyka, A.; Bauerdick, S.; Albrecht, T.; Kim, M. J.; Jemth, P.; del, J. B. E. Single-Molecule Studies of Intrinsically Disordered Proteins Using Solid-State Nanopores. Anal. Chem. 2013, 85, 2449−2456. (24) AlQuraishi, M.; McAdams, H. H. Direct Inference of Protein− DNA Interactions Using Compressed Sensing Methods. Proc. Natl. Acad. Sci. 2011, 108, 14819−14824. (25) Nakatsuji, N. Mesoscopic Science, Where Materials Become Life and Life Inspires Materials. A Great Opportunity to Push Back the



CONCLUSIONS By means of the Kirkwood-Shumaker theory, we have demonstrated the importance of the charge-regulation mechanism and the ion-dipole interaction in the complexation of proteins-charged nanoparticles at different ionic strenghs. Although the dominating term is often the charge−induced charge interaction in the low salt limit, the increase of salt concentration tends to raise the protein dipole moment and to more severely screen the charge regulation term. Therefore, at intermediate and high ionic strengths, the complexation is driven by the charge-dipole effects. These results provide a theoretical physical foundation to interpret the complexation at the wrong side of pI. They also confirm that the Kirkwood-Shumaker theory can be used to predict the complexation, and as such, it can be used to design the experimental conditions (NP charge and radius, pH, and salt concentration) for the selective bioseparation and other practical applications.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (F.L.B.S.) Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) for support via an initiation grant (Dbr: IB2013-5229). M.B. and C.P. acknowledge support from VR (Contract No. C0485101), STEM (Contract No. 34138-1) and the Research Council of Norway (Project: 221469/F20). F.L.B.S. is also thankful for the support from Fapesp (2010/50425-0), Capes (2405/13-0), and the University of São Paulo through the NAP-CatSinQ (Research Core in Catalysis and Chemical Synthesis).



REFERENCES

(1) de Vries, R.; Stuart, M. C. Theory and Simulations of Macroion Complexation. Curr. Opin. Colloid Interface Sci. 2006, 11, 295−301. (2) de Vos, W. M.; Biesheuvel, P. M.; de Keizer, A.; Kleijn, J. M.; Cohen Stuart, M. A. Adsorption of the Protein Bovine Serum Albumin in a Planar Poly(acrylic Acid) Brush Layer As Measured by Optical Reflectometry. Langmuir 2008, 24, 6575−6584. (3) Lynch, L.; Dawson, K. A. Protein-Nanoparticle Interactions. Nano Today 2008, 3, 40−47. 4082

dx.doi.org/10.1021/la500027f | Langmuir 2014, 30, 4078−4083

Langmuir

Article

Frontiers of Life, Materials, And Biomaterials Sciences. Biomater. Sci. 2013, 1, 9+. (26) de Vos, W. M.; Leermakers, F. A.; de Keizer, A.; Cohen Stuart, M. A.; Kleijn, M. M. Field Theoretical Analysis of Driving Forces for the Uptake of Proteins by Like-Charged Polyelectrolyte Brushes: Effects of Charge Regulation and Patchiness. Langmuir 2010, 26, 249− 259. (27) Kizilay, E.; Dinsmore, A. D.; Hoagland, D. A.; Sun, L.; Dubin, P. L. Evolution of Hierarchical Structures in Polyelectrolyte-Micelle Coacervates. Soft Matter 2013, 9, 7320−7332. (28) Kirkwood, J. G.; Shumaker, J. B. Forces Between Protein Molecules in Solution Arising from Fluctuations in Proton Charge and Configuration. Proc. Natl. Acad. Sci. U.S.A. 1952, 38, 863−871. (29) da Silva, F. L. B.; Lund, M.; Jönsson, B.; Åkesson, T. On the Complexation of Proteins and Polyelectrolytes. J. Phys. Chem. B 2006, 110, 4459−4464. (30) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank. Nucleic Acids Res. 2000, 28, 235−242. (31) Kesvatera, T.; Jönsson, B.; Thulin, E.; Linse, S. Ionization Behavior of Acidic Residues in Calbindin D9k. Proteins: Struct., Funct., Genet. 1999, 37, 106−115. (32) Nozaki, Y.; Tanford, C. Examination of Titratation Behavior. Methods Enzymol. 1967, 11, 715−734. (33) Levesque, D.; Weis, J. J.; Hansen, J. P. Simulation of Classical Fluids. Monte Carlo Methods in Statistical Physics, Berlin, 1986; pp 47− 119. (34) Frenkel, D.; Smit, B. Understanding Molecular Simulation: From Algorithms to Applications; Academic Press: San Diego, 1996. (35) Gottschalk, M.; Nilsson, H.; Roos, H.; Halle, B. Protein SelfAssociation in Solution: The Bovine β-Lactoglobulin Dimer and Octamer. Protein Sci. 2003, 12, 2404−2411. (36) Fogolari, F.; Ragona, L.; Licciardi, S.; Romagnoli, S.; Michelutti, R.; Ugnolini, R.; Molilari, H. Electrostatic Properties of Bovine βLactoglobulin. Proteins: Struct., Funct., Genet. 2000, 39, 317−330.

4083

dx.doi.org/10.1021/la500027f | Langmuir 2014, 30, 4078−4083