Mechanism Of Protein Adsorption Research Articles

Interfacial adsorption mechanism of FFIBP Ice-binding protein with gold nanoparticle surface (111): insights from atomistic simulations

Interfacial adsorption mechanism of FFIBP Ice-binding protein with gold nanoparticle surface (111): insights from atomistic simulations

Acidic pH impact on BSA domains I & II adsorption to a silica dioxide surface.

Bovine Serum Albumin (BSA) is a protein used as a passive blocker, adsorbing to various surfaces to reduce nonspecific interactions. BSA contains three structurally homologous domains but comprised of a different number of basic/acidic amino acids, causing each domain to respond to pH changes differently. At neutral pH, domain I contains many negatively charged residues producing a total net charge of −17. At pH 5.2, these residues become protonated, shifting the net charge to +1. The change in pH alters the charge of BSA and therefore may affect protein-surface interactions. Silica is a common surface used in bead-based immunoassays. Experimental observations suggest that adsorption occurs when both BSA and silica are negatively charged but the mechanism is not fully understood. By using theoretical techniques like molecular dynamics (MD) simulations we can analyze this protein-surface interaction and begin to understand it on a molecular level. In this study, we investigated how the change in pH can impact the adsorption of BSA to silica using MD simulations. BSA binding to silica at pH 5.2 and pH 7.0 were compared by performing simulations using NAMD. We observed that at pH 5.2, domains I and II orient closer to the surface to create many intermolecular interactions, while at pH 7.0 only domain II interacted with the silica surface. This interaction could be due to the change in surface charge of domain I at pH 7.0 and pH 5.2. At pH 7.0 domain I is highly negatively charged impacting the electrostatic interactions that are observed at pH 5.2, where domain I is less negatively charged. With MD simulations, we found pH impacts protein-surface interaction and can affect the mechanism of protein adsorption.

Protein adsorption by nanomechanical mass spectrometry: Beyond the real-time molecular weighting.

During past decades, enormous progress in understanding the mechanisms of the intermolecular interactions between the protein and surface at the single-molecule level has been achieved. These advances could only be possible by the ongoing development of highly sophisticated experimental methods such as atomic force microscopy, optical microscopy, surface plasmon resonance, ellipsometry, quartz crystal microbalance, conventional mass spectrometry, and, more recently, the nanomechanical systems. Here, we highlight the main findings of recent studies on the label-free single-molecule (protein) detection by nanomechanical systems including those focusing on the protein adsorption on various substrate surfaces. Since the nanomechanical techniques are capable of detecting and manipulating proteins even at the single-molecule level, therefore, they are expected to open a new way of studying the dynamics of protein functions. It is noteworthy that, in contrast to other experimental methods, where only given protein properties like molecular weight or protein stiffness can be determined, the nanomechanical systems enable a real-time measurement of the multiple protein properties (e.g., mass, stiffness, and/or generated surface stress), making them suitable for the study of protein adsorption mechanisms. Moreover, we also discuss the possible future trends in label-free detection and analysis of dynamics of protein complexes with these nanomechanical systems.

Biofunctional impact of textured coatings in the application of heart assist therapy

Due to a lack of organs, cardiac support systems are being implanted in patients with severe congestive heart failure. One of the solutions to overcome complications such as inflow obstruction or pump thrombosis, which may occur in the case of ventricular assist devices, is to modify the surface of cannulas for the controlled blood clotting process. The results obtained up till now for developed surface coatings clearly show the influence of topographical and mechanical parameters of the coatings on cell viability and protein adsorption mechanism. The new coatings should enable the controlled growth of scar tissue, resulting in the limitation of thromboembolic events, and the reduction of cystic tissue growth into the flow lumen. The aim of this study is to evaluate the correlation between surface topography parameters on the susceptibility of cells to grow and adhere to the substrate as a solution with potential for use in MCS (mechanical circulatory support) devices. Research on surfaces used in MCS devices and on inflow cannulas has been carried out for many years, while the novelty of the present solution makes it a milestone within that type of application simultaneously allowing for appropriate selection of process parameters. Surface modification of titanium alloy Ti6Al7Nb was carried out using vacuum powder sintering of CP-Ti (commercially pure titanium) powder with two morphologies (regular spheres and irregular grains). The characterization of coatings obtained with the proposed method and the influence of measured topographic parameters (applying scanning electron microscopy, contact angle measurement and contact profilometry) on the cytotoxicity and susceptibility to protein adsorption were presented. Advanced albumin adsorption studies have fully confirmed the dependence of surface complexity on protein adsorption. The obtained results show a high potential of the produced coatings toward enabling permanent integration at the implant with the soft tissue.

Influence of the properties of different graphene-based nanomaterials dispersed in polycaprolactone membranes on astrocytic differentiation

Composites of polymer and graphene-based nanomaterials (GBNs) combine easy processing onto porous 3D membrane geometries due to the polymer and cellular differentiation stimuli due to GBNs fillers. Aiming to step forward to the clinical application of polymer/GBNs composites, this study performs a systematic and detailed comparative analysis of the influence of the properties of four different GBNs: (i) graphene oxide obtained from graphite chemically processes (GO); (ii) reduced graphene oxide (rGO); (iii) multilayered graphene produced by mechanical exfoliation method (Gmec); and (iv) low-oxidized graphene via anodic exfoliation (Ganodic); dispersed in polycaprolactone (PCL) porous membranes to induce astrocytic differentiation. PCL/GBN flat membranes were fabricated by phase inversion technique and broadly characterized in morphology and topography, chemical structure, hydrophilicity, protein adsorption, and electrical properties. Cellular assays with rat C6 glioma cells, as model for cell-specific astrocytes, were performed. Remarkably, low GBN loading (0.67 wt%) caused an important difference in the response of the C6 differentiation among PCL/GBN membranes. PCL/rGO and PCL/GO membranes presented the highest biomolecule markers for astrocyte differentiation. Our results pointed to the chemical structural defects in rGO and GO nanomaterials and the protein adsorption mechanisms as the most plausible cause conferring distinctive properties to PCL/GBN membranes for the promotion of astrocytic differentiation. Overall, our systematic comparative study provides generalizable conclusions and new evidences to discern the role of GBNs features for future research on 3D PCL/graphene composite hollow fiber membranes for in vitro neural models.

Protein corona of SiO2 nanoparticles with grafted thermoresponsive copolymers: Calorimetric insights on factors affecting entropy vs. enthalpy-driven associations

The mechanism of protein adsorption on various surfaces of tailored functionality is often accessed by isothermal titration calorimetry (ITC), the powerful tool yielding the full set of thermodynamic parameters in one label-free experiment. In this work, the non-ionic polymer brushes with thermo-switchable hydrophilic-hydrophobic balance, based on diethylene glycol methacrylate (DEGMA) and 4-vinyl pyridine (4VP) copolymers, grafted to SiO2 nanoparticles, were titrated with individual blood proteins, their mixture, and diluted human plasma. The concentration of adsorption sites was calculated from the sizes of protein molecules, assuming the formation of a monolayer. For the titrations with the protein mixture and diluted plasma, the concentration of titrant was an estimate, limiting the accuracy of thermodynamic parameters. The particles with grafted polymer brushes showed negative ξ-potentials similar to the blood proteins and their interactions occurred against the Coulomb forces. Two cases of exothermic hydrophobic and endothermic polar interactions were distinguished. The strength of adsorption, expressed with the affinity constant Ka, was dependent on brush composition and temperature. The adsorption of proteins from blood plasma was always exothermic and, therefore, the dominating hydrophobic interactions were assumed.

Molecular dynamics study on adsorption and desorption of lysozyme above polymer antifouling membranes

Due to the lack of molecular understanding of the adsorption mechanism of protein on the surface of antifouling materials’ surface, the rational design and optimization of antifouling coatings are limited. In this work, a series of molecular dynamics simulations were conducted to study the adsorption and desorption of lysozyme above three kinds of antifouling polymer membranes, i.e. poly(sulfobetaine methacrylate) (T4-SB), poly(3-(methacryloyloxy)propane-1-sulfonate) (T4-SP), and poly(2-(dimethylamino)ethyl methacrylate) (T4-DM) grafted on polysiloxane membranes. The adsorption configuration, adsorption process, binding sites and energy, stability after adsorption and desorption ability were analyzed to reveal the adsorption and desorption mechanisms. The following conclusions were derived. The adsorption of lysozyme above T4-SB was the hardest, which is consistent with our previous work that the strongest hydration layer above T4-SB served as a physical and energy barrier to protein adsorption. The interaction between lysozyme and antifouling membranes after adsorption follows the ranking of T4-SP > T4-SB > T4-DM. The structure of lysozyme was not changed during adsorption onto all antifouling surfaces, except that the binding areas were slightly affected. The desorption ability of lysozyme above T4-DM was the worst as it adsorbed with the largest radius parallel to the surface.

Orientation and Conformation of Hydrophobin at the Oil–Water Interface: Insights from Molecular Dynamics Simulations

Hydrophobins, a new class of potential protein emulsifiers, have been extensively employed in the food, pharmaceutical, and chemical industries. However, the knowledge of the underlying molecular mechanism of protein adsorption at the oil-water interface remains elusive. In this study, all-atom molecular dynamics simulations were performed to probe the adsorption orientation and conformation change of class II hydrophobin HFBI at the cyclohexane-water interface. It was proposed that a hydrophobic dipole of the protein could be used to quantitatively predict the orientation of the adsorbed HFBI. Simulation results revealed that HFBI adsorbed at the interface with the patch-up orientation toward the oil phase, regardless of its initial orientations. HFBI's secondary structure was maintained to be intact in the course of simulations despite relatively significant variations in the tertiary structure observed, which could well preserve the bioactivity of HFBI. From the energy analysis, the driving force for interface adsorption was primarily determined by van der Waals interactions between HFBI and cyclohexane. Further analysis indicated that the adsorption orientation and conformation of HFBI at the oil-water interface were typically regulated by the hydrophobic patch and some key residues. This study provides some insights into the orientation, conformation, and adsorption mechanism of proteins at the oil-water interface and theoretical guidelines for the design and development of novel biological emulsifiers involved in the food, pharmaceutical, and chemical industries.

Enhanced Protein Adsorption Capacity of Macroporous Pectin Particles with High Specific Surface Area and an Interconnected Pore Network.

There has been much interest in developing protein adsorbents using nanostructured particles, which can be engineered porous materials with fine control of the surface and pore structures. A significant challenge in designing porous adsorbents is the high percentage of available binding sites in the pores owing to their large surface areas and interconnected pore networks. In this study, continuing the idea of using porous materials derived from natural polymers toward the goal of sustainable development, porous pectin particles are reported. The template-assisted spray drying method using calcium carbonate (CaCO3) as a template for pore formation was applied to prepare porous pectin particles. The specific surface area was controlled from 177.0 to 222.3 m2 g-1 by adjusting the CaCO3 concentration. In addition, the effects of a macroporous structure, the specific surface area, and an interconnected pore network on the protein (lysozyme) adsorption capacity and adsorption mechanism were investigated. All porous pectin particles performed rapid adsorption (∼65% total capacity within 5 min) and high adsorption capacity, increasing from 1543 to the highest value of 2621 mg g-1. The results are attributed to the high percentage of available binding sites located in the macropores owing to their large surface areas and interconnected pore networks. The macroporous particles obtained in this study showed a higher adsorption capacity (2621 mg g-1) for lysozyme than other adsorbents. Moreover, the rapid uptake and high performance of this material show its potential as an advanced adsorbent for various macromolecules in the food and pharmaceutical fields.

Albumin Protein Adsorption on CoCrMo Implant Alloy: Impact on the Corrosion Behaviour at Localized Scale

The protein adsorption and both its conformational arrangements and electrochemical interactions on the surface of metallic biomaterials has an immense impact on corrosion/biodegradation and biocompatibility of implantable metals. In this study, we used scanning Kelvin probe force microscopy (SKPFM) to reveal the synergistic effect of various bovine serum albumin (BSA) concentrations and overpotential conditions on BSA protein adsorption mechanisms and its influence on the corrosion behaviour of the CoCrMo alloy in phosphate-buffered saline solution. Electrochemical measurements showed that CoCrMo alloy was more resistant to corrosion in the 2 g l−1 BSA protein medium than in the 0.5 g l−1 one. The SKPFM analysis revealed a lower surface potential on the regions where BSA was adsorbed forming clusters, than on the un-covered CoCrMo substrate. When the surface overpotential and the protein concentration were increased from the OCP to +300 mV vs Ag/AgCl and from 0.5 to 2 g l−1, respectively, on both protein covering and surface potential were increased. Field emission scanning electron microscopy indicated that localized corrosion eventually occurred at the BSA protein/substrate interface owing to the adsorption of counterions and the difference between the surface potential values.

Role of Sporopollenin Shell Interfacial Properties in Protein Adsorption.

Sporopollenin shells isolated from natural pollen grains have received attention in translational and applied research in diverse fields of drug delivery, vaccine delivery, and wastewater remediation. However, little is known about the sporopollenin shell's potential as an adsorbent. Herein, we have isolated sporopollenin shells from four structurally diverse pollen species, black walnut, marsh elder, mugwort, and silver birch, to study protein adsorption onto sporopollenin shells. We investigated three major interfacial properties, surface area, surface functional groups, and surface charge, to elucidate the mechanism of protein adsorption onto sporopollenin shells. We showed that sporopollenin shells have a moderate specific surface area (<12 m2/g). Phosphoric acid and potassium hydroxide treatments that were used to isolate sporopollenin shells from natural pollen grains also result in the functionalization of sporopollenin shell surfaces with ionizable groups of carboxylic acid and carboxylate salt. As a result, sporopollenin shells exhibit a negative ζ potential in the range of -75 to -82 mV at pH 10 when dispersed in water. The ζ potentials of sporopollenin shells remain negative in the pH range of 2.5-11, with the absolute value of ζ potential showing a decrease with the decrease in pH. The negative surface charge promotes the adsorption of protein onto the sporopollenin shell via electrostatic interaction. Despite having a moderate surface area, sporopollenin shells adsorb a significant amount of lysozyme (145-340 μg lysozyme per mg of sporopollenin shells). Lysozyme adsorption onto sporopollenin shells alters the surface, and the surface charge becomes positive at acidic pH. Overall, this study demonstrates the potential of sporopollenin shells to adsorb proteins, highlights the critical role of sporopollenin shell's interfacial properties in protein adsorption, and identifies the mechanism of protein adsorption on sporopollenin shells.

Effect of Protein Adsorption on Air Plastron Behavior of a Superhydrophobic Surface.

Protein fouling on critical biointerfaces causes significant public health and clinical ramifications. Multiple strategies, including superhydrophobic (SHP) surfaces and coatings, have been explored to mitigate protein adsorption on solid surfaces. SHP materials with underwater air plastron (AP) layers hold great promise by physically reducing the contact area between a substrate and protein molecules. However, sustaining AP stability or lifetime is crucial in determining the durability and long-term applications of SHP materials. This work investigated the effect of protein on the AP stability using model SHP substrates, which were prepared from a mixture of silica nanoparticles and epoxy. The AP stability was determined using a submersion test with real-time visualization. The results showed that AP stability was significantly weakened by protein solutions compared to water, which could be attributed to the surface tension of protein solutions and protein adsorption on SHP substrates. The results were further examined to reveal the correlation between protein fouling and accelerated AP dissipation on SHP materials by confocal fluorescent imaging, surface energy measurement, and surface robustness modeling of the Cassie-Baxter to Wenzel transition. The study reveals fundamental protein adsorption mechanisms on SHP materials, which could guide future SHP material design to better mitigate protein fouling on critical biointerfaces.

Adsorption of Mussel Protein on Polymer Antifouling Membranes: A Molecular Dynamics Study.

Biofouling is one of the most difficult problems in the field of marine engineering. In this work, molecular dynamics simulation was used to study the adsorption process of mussel protein on the surface of two antifouling films—hydrophilic film and hydrophobic film—trying to reveal the mechanism of protein adsorption and the antifouling mechanism of materials at the molecular level. The simulated conclusion is helpful to design and find new antifouling coatings for the experiments in the future.

Kinetic investigation of protein adsorption into polyelectrolyte brushes by quartz crystal microbalance with dissipation: The implication of the chromatographic mechanism

With the growing concerns of polymer-grafted ion-exchange chromatography, the importance of protein adsorption on charged polymer-grafted surfaces cannot be stressed enough. However, a full understanding in adsorption in polymer brushes is still a great challenge due to the lack of in situ characterization technique. In this work, we use quartz crystal microbalance with dissipation to in situ investigate adsorption kinetics of γ-globulin and recombinant human lactoferrin on poly(3-sulfopropyl methacrylate) (pSPM) sensors prepared via atom transfer radical polymerization. With an increase of chain length and grafting density, great increasing amounts of proteins on pSPM-grafted sensors revealed that protein underwent a transition from monolayer to multilayer adsorption. It was attributed to direct protein binding into charged brushes, in which more binding sites involved and more coupled water lost. However, such a strong binding and rigid structure of proteins limited the protein transport in pSPM brushes and “chain delivery” effect. With an increase in grafting density, moreover, denser brushes hindered adjustment in protein conformation in pSPM brushes and further exacerbated protein transport in pSPM brushes. Furthermore, the influence of buffer pH and salt concentration further validated the ion exchange characteristics of protein adsorption into pSPM brushes. The research provided a variety of in situ evidence of protein binding and conformation evolution in pSPM brushes and elucidated mechanism of protein adsorption in pSPM brushes.

Effect of Branching Degree of Dendritic Polyglycerols on Plasma Protein Adsorption: Relationship between Hydration States and Surface Morphology.

This study focuses on dendritic glycerols and investigates the construction of biocompatible surfaces by understanding how differences in the branching of these molecules change the interactions with the biological components. The two molecules, polyglycerol dendrimer (PGD), which has a completely branched structure, and hyperbranched polyglycerol (HPG), which has an incompletely branched structure, are compared and the differences in branching are evaluated. It is shown that PGD has a little bit more intermediate water than HPG, which reflects the differences in the branching. The effect of surface state on the adsorption of the plasma proteins, human serum albumin (HSA), fibrinogen (Fib), and fibronectin (FN), is discussed by modifying a glass surface using these molecules with different hydration states. The adsorption of HSA decreases to several percent for HPG and 10% for PGD compared to unmodified substrate. Although the adsorption of Fib decreases to 5% for HPG, an increase to 150% is observed for PGD. Since this specific Fib adsorption observed only onto PGD is suppressed in the cases of a mixed solution of HSA and Fib or sequentially using HSA solution and then Fib solution, it is thought that the Vroman effect is suppressed on the PGD-modified surface. Furthermore, when AFM measurements are performed in PBS to understand the surface roughness, PGD is found to be more highly non-uniform. Because of this, the nanometer scale roughness that is significantly observed only on the PGD-modified surface is thought to have an effect on the characteristic adsorption properties of Fib. Thus, although both PGD and HPG with different branching have intermediate water, the proportion differs between PGD and HPG. Therefore, it is found that differences occur in the plasma protein adsorption mechanisms depending on the coordinates and density of hydroxyl groups within the molecules.

Bioinspired polysaccharide hybrid hydrogel promoted recruitment and chondrogenic differentiation of bone marrow mesenchymal stem cells

Cartilage regeneration by biomimetic cartilage matrix with synchronously recruited stem cells was one of ideal strategies. Inspired by catechol for proteins adhesion, dopamine modified polysaccharide hybrid hydrogel (HD-C) was prepared by integrating collagen I (Col I) and hyaluronic acid derivatives (HA-DN) with sulfhydryl modified polysaccharide hybrid hydrogel (HS-C) as control. Because of double-crosslinking architecture, HD-C hydrogel was endowed with a more compact pore structure, higher mechanical properties and water retention ability in comparison with those of HS-C hydrogel. Meanwhile, it significantly promoted the proliferation and spread of rabbit bone marrow stem cells (rBMSCs), and accelerated cartilaginous matrix secretion. RT-PCR results also verified higher related gene expression of chondrogenesis (Sox 9, Agg and Col II). Moreover, HD-C hydrogel could enhance the enrichment and migration of rBMSCs in vitro by potential functional protein adsorption mechanisms, and this phenomenon was further confirmed by more rBMSCs migration in short-term joint implantation experiments in vivo.

Influence of the second phase on protein adsorption on biodegradable Mg alloys’ surfaces: Comparative experimental and molecular dynamics simulation studies

The effect of the second phase on the mechanical properties and corrosion resistance of Mg alloys has been systematically studied. However, there is limited information on the effect of the second phase on protein adsorption behavior. In the present study, the effect of the second phase on protein adsorption on the surfaces of biodegradable Mg alloys was investigated using experimental methods and molecular dynamics (MD) simulations. The experimental results showed that the effect of the second phase on fibrinogen adsorption was type-dependent. Fibrinogen preferentially adsorbed on Y-, Ce-, or Nd-involved second phases, while the second phase containing Zn inhibited its adsorption. MD simulations revealed the mechanism of the second phase that influenced protein adsorption in terms of charge distribution, surface-protein interaction energy, and water molecule distribution. Our studies proposed a deep understanding of the design of Mg-based biomaterials with superior biocompatibility. Statement of SignificanceMechanical properties, uniform degradation, and biocompatibility must be considered while designing biomedical Mg alloys. To improve the mechanical properties and corrosion resistance of Mg alloys, the second phase is usually required. However, the effects of the second phase on biocompatibility of Mg alloys have been rarely reported. Here, the influence of the second phase on protein adsorption was experimentally studied by designing Mg alloys with different types of second phase. The first principle calculation and MD simulation were used to reveal the mechanism by which the second phase influences protein adsorption. This work could be used to better elucidate the protein adsorption mechanisms and design principles to improve the biocompatibility of Mg alloys.

Effect of pore structure on protein adsorption mechanism on ion exchange media: A preliminary study using low field nuclear magnetic resonance

The adsorption process of bovine serum albumin (BSA), ovalbumin (OVA) and human immunoglobulin G (IgG) on agarose ion-exchange media Q Sepharose FF and two dextran-grafted agarose media including Q Sepharose XL and Capto Q were studied using low field nuclear magnetic resonance (NMR). The T2 relaxation time was found directly proportional to the pore size and diminished after protein adsorbed, therefore, a theoretical model describing the relationship between protein binding amount and T2 relaxation signals was established. The model parameters, a, which reflects the contact area between the adsorbed protein and media surface, and the δ, which defined as the ratio of the protein volume to the pore volume after adsorption, were found to describe the pore occupation states of proteins in media with different pore structures very well. For small proteins, such as BSA and OVA, monolayer adsorption occurred on Q Sepharose FF, which has no dextran chains. Therefore, the adsorbed protein only occupied 49.05% of the pore volume for BSA and 25.51% for OVA, and contact area of each protein on the media were also low, suggesting mostly monolayer adsorption occurred. In the contrast, their adsorption to Q Sepharose XL and Capto Q with dextran chains tended to form multilayer adsorption, thus higher contact area was obtained and the pore volumes were almost 100% occupied. For large protein, such as IgG, the adsorption to all these three media was similar and about 30% of the pore volume were occupied, probably due to the similar restriction for IgG to entering the media pore. Results of this study will help to elucidate the relationship between protein adsorption and pore size variation, which present the significance of low field NMR in understanding protein adsorption mechanism.

Study on Adsorption Conditions of Corn Protein and Starch

This paper mainly measured the adsorption of corn protein on corn starch, studied the changes of protein adsorption under different conditions, and discussed the adsorption mechanism of corn starch and protein. The results showed that when the adsorption time was 1h, the amount of adsorbed protein was the largest. Heating will increase the adsorption capacity; In the range of pH3-9, with the increase of pH value, the adsorption amount of protein firstly increases and then decreases, and the adsorption amount is the largest when pH value is about 6. Adsorption decreases with the increase of ionic strength. The addition of acid pulp can reduce the occurrence of protein adsorption, and the amount of protein adsorption is the least when the acid pulp is added to 50%. Increasing the number of pores and pore size on the surface of starch particles can increase protein adsorption. The adsorption isotherm of corn protein used in this experiment on corn starch granules is S-type, belonging to type II adsorption isotherm.

Adsorption of Myoglobin and Corona Formation on Silica Nanoparticles.

The adsorption of proteins from aqueous medium leads to the formation of protein corona on nanoparticles. The formation of protein corona is governed by a complex interplay of protein–particle and protein–protein interactions, such as electrostatics, van der Waals, hydrophobic, hydrogen bonding, and solvation. The experimental parameters influencing these interactions, and thus governing the protein corona formation on nanoparticles, are currently poorly understood. This lack of understanding is due to the complexity in the surface charge distribution and anisotropic shape of the protein molecules. Here, we investigate the effect of pH and salinity on the characteristics of corona formed by myoglobin on silica nanoparticles. We experimentally measure and theoretically model the adsorption isotherms of myoglobin binding to silica nanoparticles. By combining adsorption studies with surface electrostatic mapping of myoglobin, we demonstrate that a monolayered hard corona is formed in low salinity dispersions, which transforms into a multilayered hard + soft corona upon the addition of salt. We attribute the observed changes in protein adsorption behavior with increasing pH and salinity to the change in electrostatic interactions and surface charge regulation effects. This study provides insights into the mechanism of protein adsorption and corona formation on nanoparticles, which would guide future studies on optimizing nanoparticle design for maximum functional benefits and minimum toxicity.