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Competitive Protein Adsorption on Charge Regulating Silica-Like Surfaces: The Role of Protonation Equilibrium

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Journal of Physics: Condensed Matter
Authors:
Marilina Cathcarth at National University of La Plata
Marilina Cathcarth
Agustin S Picco at National University of La Plata
Agustin S Picco
Gabriela Mondo at State University of Campinas (UNICAMP)
Gabriela Mondo
Mateus B Cardoso
Mateus B Cardoso
Mateus B Cardoso
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Abstract and Figures

We develop a molecular thermodynamic theory to study the interaction of some proteins with a charge regulating silica-like surface under a wide range of conditions, including pH, salt concentration and protein concentration. Proteins are modeled using their three dimensional structure from crystallographic data and the average experimental pKa of amino acid residues. As model systems, we study single-protein and binary solutions of cytochrome c, green fluorescent protein (GFP), lysozyme and myoglobin. Our results show that protonation equilibrium plays a critical role in the interactions of proteins with these type of surfaces. The terminal hydroxyl groups on the surface display considerable extent of charge regulation; protein residues with titratable side chains increase protonation according to changes in the local environment and the drop in pH near the surface. This behavior defines protein-surface interactions and leads to the emergence of several phenomena: (i) a complex non-ideal surface charge behavior; (ii) a non-monotonic adsorption of proteins as a function of pH; and (iii) the presence of two spatial regions, a protein-rich and a protein-depleted layer, that occur simultaneously at different distances from the surface when pH is slightly above the isoelectric point of the protein. In binary mixtures, protein adsorption and surface-protein interactions cannot be predicted from single-protein solution considerations.
Schematic of the system under study: a planar surface having exposed hydroxyl groups is in contact with an aqueous protein solution; the surface density of these units is σ OH. A fraction fO− of these groups is dissociated (negatively charged). Coordinate z measures the distance from the surface. Far from the surface (z → ∞), the composition of the bulk solution is controlled, including its pH, concentration of salt and that of each protein. We consider mono-protein and binary solutions of cytochrome c, green fluorescent protein (GFP), lysozyme, and myoglobin.
Schematic of the system under study: a planar surface having exposed hydroxyl groups is in contact with an aqueous protein solution; the surface density of these units is σ OH. A fraction fO− of these groups is dissociated (negatively charged). Coordinate z measures the distance from the surface. Far from the surface (z → ∞), the composition of the bulk solution is controlled, including its pH, concentration of salt and that of each protein. We consider mono-protein and binary solutions of cytochrome c, green fluorescent protein (GFP), lysozyme, and myoglobin.
… 
Schematic of the coarse-grained molecular model used to represent cytochrome c, GFP, myoglobin and lysozyme. These proteins are described using its three-dimensional structure obtained from the protein data bank [50]. Each amino acid residue is modeled as a single particle. (A) 3D representation of the proteins in our coarse-grained model, where each residue has been colored according to the protonation behavior of its side-chain: either acidic, basic or charge neutral. (B) Plot of the charge number of these proteins in a dilute bulk solution, Q bulk, as a function of pH.
Schematic of the coarse-grained molecular model used to represent cytochrome c, GFP, myoglobin and lysozyme. These proteins are described using its three-dimensional structure obtained from the protein data bank [50]. Each amino acid residue is modeled as a single particle. (A) 3D representation of the proteins in our coarse-grained model, where each residue has been colored according to the protonation behavior of its side-chain: either acidic, basic or charge neutral. (B) Plot of the charge number of these proteins in a dilute bulk solution, Q bulk, as a function of pH.
… 
Plots of surface charge density, Q s, as a function of pH. Panel (A) shows surfaces having different densities of terminal hydroxyl groups, σ OH, for 10 mM NaCl. The lower-left inset displays the average degree of charge of hydroxyl groups on the surface, fO− , at the same conditions as the main plot; the dashed-line curve represents the dissociation of these groups under ideal conditions. Panel (B) presents a σ OH = 4.6 nm⁻² surface in contact with solutions having different salt concentrations; the dashed-line curve is the expected surface charge under the ideal dissociation assumption. pK OH = 6.5 in all calculations.
Plots of surface charge density, Q s, as a function of pH. Panel (A) shows surfaces having different densities of terminal hydroxyl groups, σ OH, for 10 mM NaCl. The lower-left inset displays the average degree of charge of hydroxyl groups on the surface, fO− , at the same conditions as the main plot; the dashed-line curve represents the dissociation of these groups under ideal conditions. Panel (B) presents a σ OH = 4.6 nm⁻² surface in contact with solutions having different salt concentrations; the dashed-line curve is the expected surface charge under the ideal dissociation assumption. pK OH = 6.5 in all calculations.
… 
(A) Plots of the adsorption, Γ, as a function of pH for different proteins in 1 μM concentration; each panel corresponds to a particular salt concentration. (B) Plots showing Γ as a function of protein concentration at pH 7.
(A) Plots of the adsorption, Γ, as a function of pH for different proteins in 1 μM concentration; each panel corresponds to a particular salt concentration. (B) Plots showing Γ as a function of protein concentration at pH 7.
… 
(A) Plot of the potential of mean force acting on a GFP as a function of the distance z between the center of mass of the protein and the surface. The various curves correspond to different pH values around the intrinsic pI of the protein; the inset shows the corresponding adsorption, where these pH values are marked. (B) Ensemble average charge number of a GFP protein with center of mass at z, at the same conditions of panel (A). All results correspond to 1 μM GFP and 10 mM NaCl.
+4
(A) Plot of the potential of mean force acting on a GFP as a function of the distance z between the center of mass of the protein and the surface. The various curves correspond to different pH values around the intrinsic pI of the protein; the inset shows the corresponding adsorption, where these pH values are marked. (B) Ensemble average charge number of a GFP protein with center of mass at z, at the same conditions of panel (A). All results correspond to 1 μM GFP and 10 mM NaCl.
… 
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Journal of Physics: Condensed Matter
J. Phys.: Condens. Matter 34 (2022) 364001 (13pp) https://doi.org/10.1088/1361-648X/ac6388
Competitive protein adsorption on charge
regulating silica-like surfaces: the role
of protonation equilibrium
Marilina Cathcarth1, Agustin S Picco1, Gabriela B Mondo2,3,
Mateus B Cardoso2,3and Gabriel S Longo1,∗
1Instituto de Investigaciones Fisicoquímicas, Te´
oricas y Aplicadas (INIFTA), UNLP-CONICET, La Plata,
Argentina
2Brazilian Synchrotron (LNLS) and Brazilian Nanotechnology Laboratory (LNNano),
Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, Brazil
3Institute of Chemistry (IQ), University of Campinas (UNICAMP), Campinas, Brazil
E-mail: longogs@inifta.unlp.edu.ar
Received 31 December 2021, revised 16 February 2022
Accepted for publication 1 April 2022
Published 5 July 2022
Abstract
We develop a molecular thermodynamic theory to study the interaction of some proteins with
a charge regulating silica-like surface under a wide range of conditions, including pH, salt
concentration and protein concentration. Proteins are modeled using their three dimensional
structure from crystallographic data and the average experimental pKa of amino acid residues.
As model systems, we study single-protein and binary solutions of cytochrome c, green
uorescent protein, lysozyme and myoglobin. Our results show that protonation equilibrium
plays a critical role in the interactions of proteins with these type of surfaces. The terminal
hydroxyl groups on the surface display considerable extent of charge regulation; protein
residues with titratable side chains increase protonation according to changes in the local
environment and the drop in pH near the surface. This behavior denes protein–surface
interactions and leads to the emergence of several phenomena: (i) a complex non-ideal surface
charge behavior; (ii) a non-monotonic adsorption of proteins as a function of pH; and (iii) the
presence of two spatial regions, a protein-rich and a protein-depleted layer, that occur
simultaneously at different distances from the surface when pH is slightly above the isoelectric
point of the protein. In binary mixtures, protein adsorption and surface–protein interactions
cannot be predicted from single-protein solution considerations.
Keywords: silica surface, protein adsorption, protonation, charge regulation, molecular theory
SSupplementary material for this article is available online
(Some gures may appear in colour only in the online journal)
1. Introduction
Understanding protein adsorption onto nanomaterial surfaces
is currently a problem of key relevance in a variety of biomed-
ical applications, including controlled release, drug delivery,
bioimaging, implant technology, among others [1,2]. Upon
exposure to uids containing proteins (plasma, tears, and cell
∗Author to whom any correspondence should be addressed.
culture media to name a few examples) both nanoparticles [3]
and macroscopic substrates [4] tend to adsorb them. Hence,
the properties of the nanomaterial surface are deeply modied,
altering the way it interacts with the biological components
[1,5]. As a result, non-specic adsorption of proteins must
be prevented in many applications [6,7]. Protein corona can
negatively affect on the targeting capability of nanomaterials
[8]. On the contrary, the adsorption of some specic protein
1361-648X/22/364001+13$33.00 1 ©2022 IOP Publishing Ltd Printed in the UK
... In one of the many examples of Argentine-Brazilian collaboration, Cathcarth et al [28] developed a molecular thermodynamic theory to investigate the adsorption of proteins (cytochrome c, green fluorescent protein, lysozyme, and myoglobin) onto a charge-regulating silica-like surface. Their study encompasses a wide range of experimental conditions, including pH, salt, and protein concentrations. ...
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