Proteins at the air-water interface play crucial roles in many scenarios
a Biological and technological relevance of proteins at the AWI
The surface properties of proteins are crucial in material science, food science, and medical science Research has concentrated on protein partitioning at the lipid-water interface to elucidate signal transduction and material transport mechanisms in cells Additionally, protein adsorption at the solid-liquid interface is a key area of study aimed at creating more biocompatible materials for implants and drug delivery systems Furthermore, proteins at the air-water interface provide foundational insights into more complex lipid-water and solid-liquid interactions, with direct applications across various fields.
In material science, ranaspumin from frog nest foams has been shown to adsorb at the air-water interface (AWI), contributing to the formation of stable foams with 90% air and 10% liquid This bio-foam system holds promise for applications as biochemical reactors on micro- and nano-scales Additionally, research on spider silk proteins at the AWI has revealed their ability to self-assemble into highly elastic and extensible filaments Block copolymers mimicking silk proteins have been synthesized, demonstrating the formation of viscoelastic thin films at the AWI In food science, milk proteins such as β-casein and β-lactoglobulin are crucial for foam stability in dairy products, as they interact with surfactants at the air-water or oil-water interface In medical science, lung surfactant, a mixture of phospholipoproteins and lipids, reduces surface tension at the AWI of alveoli, and its deficiency can lead to alveolar collapse and respiratory failure.
Plasma proteins and their interactions at the air-water interface (AWI) have garnered significant attention, with at least 289 plasma proteins identified in blood plasma or serum Among these, serum albumin, fibrinogen, and immunoglobulin G are the most abundant Researchers are particularly interested in the surface properties of plasma proteins for several reasons: they provide insights into plasma protein behavior at solid surfaces, which is crucial for understanding air embolism pathogenesis linked to plasma proteins on air bubble surfaces, and they can enhance the design and sensitivity of antibody-based sensors in biotechnology Consequently, this thesis focuses on studying serum albumin, fibrinogen, and immunoglobulin G at the AWI.
Experimental methods to study proteins at the air-water interface
Tensiometry is a widely utilized technique for examining the surface properties of amphiphiles by measuring the surface tension (\( \gamma \)) at the air-liquid interface Surface tension, expressed in Newtons per meter (N/m), arises from intermolecular forces among solvent molecules and acts to minimize the liquid's surface area, thereby reducing surface energy Notably, the air-water interface (AWI) exhibits the highest surface tension in nature due to robust hydrogen bonding When amphiphiles adsorb at this interface, they disrupt the hydrogen bonds between water molecules, leading to a reduction in surface tension (\( \gamma \)) The relationship between surface tension and surfactant concentration is quantitatively described by the Gibbs isotherm.
The gas constant \( R \), temperature \( T \), surface excess \( \Gamma \) (measured in mol/m² or mg/m²), and bulk concentration \( C \) (measured in mol/m³ or mg/m³) are key parameters in understanding surface phenomena Figure 1.2 illustrates the concept of surface excess, highlighting the distinction between surface concentration and bulk concentration, which is influenced by surface tension \( \gamma \) and bulk concentration \( C \) of amphiphiles, as described by Equation 1.1 However, for proteins, the relationship between surface excess and surface tension often deviates from the Gibbs isotherm, with further explanations provided in section IIIa.
Surface pressure (\(π\)) in tensiometry refers to the difference between the surface tension of a clean solvent (\(γ_0\)) and that of a solution with adsorbed solute molecules (\(γ\)) This relationship is expressed in Eq 1.2 For aqueous solutions, \(γ_0\) is typically the surface tension of pure water at 20 °C, which is 72.8 mN/m Consequently, the adsorption of amphiphiles results in an increase in surface pressure (\(π\)).
Various types of tensiometers, such as the Du Noüy-Padday probe, Wilhelmy plate, bubble pressure tensiometer, goniometer, and Du Noüy ring, measure surface tension or pressure through distinct principles The methods utilized in this thesis are detailed in Chapters 2-4 within the "Material and Experimental Methods" section.
The Du Noüy-Padday probe, Du Noüy ring, and Wilhelmy plate methods are commonly used in conjunction with the Langmuir-Blodgett (LB) trough to measure surface pressure at varying amphiphile concentrations In the LB trough, solute molecules adsorb at the air-water interface (AWI) to form a thin layer The surface concentration of these molecules can be adjusted by altering the surface area of the two-dimensional layer through compression or relaxation, as surface concentration (\(Γ\)) is inversely related to surface area (\(A\)) By moving the barriers and simultaneously measuring surface pressure via tensiometry, one can obtain surface pressure-surface area (\(π-A\)) or surface pressure-surface concentration (\(π-Γ\)) isotherms Kinks in the isotherm typically indicate lateral phase transitions in lipid monolayers, while such shape changes are absent in protein systems due to the flexibility of protein chains.
The Langmuir trough offers a significant advantage by allowing the easy transfer of interfacial films to solid substrates for further analysis through Langmuir-Blodgett (LB) or Langmuir-Schaefer (LS) transfer methods This capability has broadened the applications of the LB trough However, a notable drawback is its high compound consumption, as it requires a large sample volume, making it less cost-effective for studying certain proteins that may be expensive or labor-intensive to acquire.
Figure 1.1 Interaction between water molecules at the air-water interface causes surface tension
The interaction among neighboring water molecules is less pronounced than in the bulk phase, resulting in increased free energy and reduced hydrogen bonding between the molecules.
Figure adapted from Kibron Inc (http://www.kibron.com/surface-tension)
Figure 1.2 Definition of surface excess
In the given diagram, the X-axis represents concentration while the Y-axis indicates the distance normal to the phase boundary The bold curved lines illustrate the concentration profiles of the solute on the left and the solvent on the right, with vertical broken lines denoting concentrations in the reference system The chain-dotted lines mark the boundaries of the interfacial layer A bold horizontal line indicates the primary dividing surface, while a dotted horizontal line represents an alternative location for this surface The surface excess is calculated as the sum of the shaded areas above and below the dividing surface Selecting the upper dividing surface results in a surface excess of zero, whereas choosing the lower surface yields a non-zero surface excess.
Figure adapted from: Mitropoulos, A C.: J Eng Sci Technol Rev 2008, 1, 1-3
Figure 1.3 Comparison of π- A isotherms of lipid and protein at the AWI
The isotherm of dipalmitoylphosphatidylcholine (DPPC) monolayer illustrates the lateral phase transitions within the DPPC film at varying surface pressures, highlighting phases such as gas, liquid-expanded (LE), liquid-condensed (LC), and the coexistence of liquid-condensed and liquid-expanded (LE–LC), along with the collapse of the interfacial film Additionally, the isotherm of β-casein reveals the structural changes occurring in the protein film.
Figure 1.3 (A) is adapted from: Wüstneck, R et al.; Adv Colloid Interface Sci 2005, 117, 33-58
Figure 1.3 (B) is adapted from: Nino, M R R et al.; Colloid Surf B-Biointerfaces 1999, 12, 161-173 b Fluorescence microscopy
Fluorescence microscopy, first utilized at the AWI in the 1980s to investigate lipid monolayer systems, has since been effectively applied to explore protein-lipid interactions This technique is particularly suited for studying protein films due to its sub-micron spatial resolution and high signal-to-noise ratio, making it ideal for imaging film morphology Additionally, its temporal resolution, ranging from milliseconds to a few seconds, allows for the observation of dynamic processes at the interface Fluorescence microscopy can be employed independently or in conjunction with other methods, such as the Langmuir trough, for in situ measurements.
The advancement of fluorescence microscopy has significantly broadened its application in studying interfacial phenomena Confocal laser scanning microscopy (CLSM) enhances the spatial resolution of widefield fluorescence microscopy by incorporating optical sectioning capabilities, which effectively eliminates out-of-focus light and reduces image degradation This sectioning ability enables researchers to image planes perpendicular to the air-water interface (AWI), allowing for semi-quantitative analysis of dye-labeled amphiphiles at the AWI through changes in fluorescence intensity.
Figure 1.4 Vertical imaging of molecules at the AWI by CLSM
To obtain a vertical fluorescence intensity profile, the following procedures are employed: a) scanning the xy plane using Confocal Laser Scanning Microscopy (CLSM); b) stacking the resulting 2D images; and c) processing the image stack to generate a fluorescence intensity profile along the z-axis This method is applied to fluorescence image stacks of FITC-labeled pullulan, which is not surface-active, and FITC-labeled cholesterol-bearing pullulan, which is surface-active, at the Air-Water Interface (AWI).
Figure adapted from: Gluck, G et al.; Chem Lett 1996, 209-210 c Atomic force microscopy
Atomic force microscopy (AFM) is an effective technique for imaging the topography of thin films with nanometer resolution, initially applied to study protein layers at the air-water interface (AWI) by Gunning et al They successfully imaged β-casein and bovine serum albumin (BSA) at the hexadecane/water interface, revealing detailed structures at the molecular level However, AFM has limitations, as transferring the film to a solid substrate is necessary for thickness measurement and morphology imaging, which can alter the film structure due to substrate type and hydration state changes Therefore, ensuring high transfer quality is essential for accurate AFM interpretation, and complementary methods are needed to verify transfer quality and compare film structures before and after transfer.
In addition to the previously mentioned methods, various other techniques have been developed and utilized to study molecules at the AWI While these approaches were not directly employed in the research projects discussed in this thesis, the findings are frequently compared to literature results obtained through different techniques Therefore, this section provides a brief overview of these additional methods to aid in data interpretation.
Interfacial sheer rheology (ISR) is a valuable technique for analyzing the flow properties of interfacial layers by measuring the storage moduli (G’) and loss moduli (G’’) This method assesses the elasticity, which reflects the strength of intermolecular forces, and viscosity, which indicates the diffusion of molecules within the layer ISR offers insights into molecular organization at the air-water interface (AWI) The technique can be categorized into macroscopic and microscopic rheology; macroscopic rheology evaluates average elasticity and viscosity, while microscopic rheology reveals heterogeneity in the 2D layer at the micrometer scale, proving particularly beneficial for studying protein films.
Protein behaviors at the air-water interface
a Adsorption, conformation and structure of protein assembly at the AWI
Unlike small-molecule surfactants, protein adsorption does not result in a simultaneous increase in surface pressure and surface excess, and their relationship does not adhere to the Gibbs isotherm This phenomenon is attributed to two unique characteristics of proteins.
The proteins in the surface layer can gradually alter their conformation at the air-water interface (AWI), resulting in a continuous change in surface pressure At high bulk concentrations, proteins may form multilayers at the interface, leading to an increase in surface excess without a corresponding change in surface pressure Consequently, surface pressure and surface excess are not directly correlated, necessitating independent measurement of these two parameters Given the significant structural and compositional diversity among proteins, it is essential to investigate the adsorption kinetics of different proteins on a case-by-case basis.
The reversibility of protein adsorption at the air-water interface (AWI) remains a topic of debate However, a slow desorption rate of proteins from this interface is frequently observed When proteins adsorb, they tend to partially unfold, exposing hydrophobic regions to minimize surface energy, which can result in structural distortion This conformational change can persist for hours or even days, as indicated by the continuous increase in surface pressure after reaching equilibrium Depending on the protein's stability, these conformational changes may lead to denaturation.
Intermolecular forces, including chemical, electrostatic, and hydrodynamic interactions, play a crucial role in driving protein molecules into assemblies and networks Rheological studies indicate that the shear viscosity of eight different protein films increases continuously over 30 hours, suggesting enhanced intermolecular cohesion or covalent bonding Structural differences among the films formed by various proteins arise from their distinct conformations and stabilities For instance, atomic force microscopy (AFM) studies reveal that the globular shape of bovine serum albumin (BSA) is maintained in the interfacial film, while a more disordered network is observed in β-casein films Imaging studies have also reported heterogeneity in protein films formed from single components, with Powers et al identifying microdomains of varying geometries in a synthetic amphiphilic peptide using fluorescent imaging Additionally, Brewster angle microscopy (BAM) has shown domain formation when globulin 11S adsorbs to the air-water interface (AWI), and microrheology has revealed mechanical heterogeneity at the micrometer scale in β-lactoglobulin films at the AWI, indicating the presence of distinct domains.
Figure 1.5 AFM images of interfacial film of BSA or β-casein transferred onto mica
(A, B) BSA prepared at the hexadecane/water interface, (C) β-casein prepared at the AWI Height in gray scale (black to white) (A) 0-7.6 nm, (B) 0-7.0 nm, (C) 0-2.7 nm Scale bar: 100 nm
Figure adapted from: Gunning, A P et al.; J Colloid Interface Sci 1996, 183, 600-602
Serum albumin, a key plasma protein, exhibits a heart-shaped structure as revealed by crystallography, although an ellipsoid shape has been traditionally used in hydrodynamic studies It was previously believed that serum albumin undergoes structural elongation upon absorption at the air-water interface, but it remains stable due to its intramolecular scaffold The extent of conformational change is influenced by the bulk concentration of serum albumin, with studies showing that higher surface coverage of bovine serum albumin (BSA) results in less structural alteration Recent microrheology research indicates that in albumin films, surface elasticity does not increase proportionally with viscosity, suggesting an annealing mechanism rather than covalent bonding among albumin molecules.
Fibrinogen can be visualized as three spheres linked by two rods, which restricts its packing density at interfaces Its aqueous solution exhibits moderate surface activity, and ellipsometry measurements reveal a high water content in fibrinogen layers at the air-water interface (AWI) Additionally, studies utilizing Brewster angle microscopy (BAM) and scanning electron microscopy (SEM) demonstrate the formation of fibrils during the organization process at the AWI.
IgG is a Y-shaped antibody that forms a monolayer at the air-water interface (AWI) even at high bulk concentrations Its orientation shifts from side-on to end-on as the surface area decreases or the bulk concentration increases Studies utilizing fluorescent antigen binding have demonstrated that IgG experiences partial unfolding after adsorption at the interface.
Table 1.1 Crystal structures and geometric dimensions of serum albumin, fibrinogen and IgG
[*] Ferrer, M L et al.; Biophys J 2001, 80, 2422-2430 [**] Hall, C E et al.; J Biophys Biochem Cytol 1959, 5, 11-16 [***] Zhao, X et al.; J R Soc., Interface 2009, 6, S659-S670 b Interactions between proteins
In biological systems, multiple protein types coexist in solution, particularly at biological interfaces where signaling proteins engage through specific recognition at the water-lipid interface For plasma proteins, competition for interfacial area is a key interaction, which involves two steps: the diffusion rate, determining how quickly proteins reach the interface, and the protein's affinity for the interface, influencing the stability of interfacial assemblies Smaller proteins typically arrive first due to their size, but can be replaced by larger proteins with higher affinity, a phenomenon known as the Vroman effect, first identified in blood protein adsorption Studies, such as those by Krishnan et al., have shown that different blood plasma and serum proteins exhibit similar concentration dependencies, making it difficult to discern specific protein compositions at the air-water interface using tensiometry alone Additionally, the mechanical properties of the interfacial film are significantly influenced by interactions among different proteins; for instance, the addition of serum albumin to IgG reduces the film's elasticity, suggesting that serum albumin disrupts intermolecular interactions within the film Overall, studying protein competition at the air-water interface presents considerable challenges.
The increasing use of synthetic polymers in biomaterials, biosensors, and drug delivery has made it crucial to understand and control the interactions between synthetic polymers and proteins at interfaces Recent advancements have introduced methods for integrating antibodies into polymeric thin films for immunosensing, highlighting the need for a deeper comprehension of protein-polymer interactions at biological interfaces.
Many copolymers act as surfactants, adsorbing at interfaces and competing with protein adsorption These surfactants significantly reduce surface tension and alter the rheological behavior of the surface layer by interacting with proteins at the air-water interface (AWI) It is suggested that surfactants displace proteins heterogeneously from the AWI, following an "orogenic" model based on previous AFM studies However, direct evidence from in situ measurements has primarily come from BAM studies, leaving certain aspects of dynamic transitions still under-explored.
Pre-spreading polymer layers at the air-water interface (AWI) can effectively modulate interfacial energy, promoting protein adsorption Enhanced protein adsorption has been achieved through the creation of superhydrophobic polymer surfaces or by incorporating specific protein ligands Recent advancements have demonstrated tunable protein adsorption on polymer films by utilizing spatially separated chemical components with varying hydrophobicity or by altering the surface charge of polymer thin films Understanding the role of polymer thin films at the AWI as templates for protein assembly can significantly expand the methods available for controlling protein adsorption and assembly.
Research scope
This thesis employs fluorescence microscopy, alongside tensiometry and AFM, to investigate plasma proteins and their interactions with polymers at the AWI, enhancing both methodological approaches and the understanding of interfacial behavior Chapter 2 focuses on the morphological study of human serum albumin (HSA) self-assembly influenced by solution conditions Chapter 3 presents a semi-quantitative analysis of the adsorption and competition between plasma proteins and the block copolymer Pluronic F-127 Chapter 4 explores the interaction between Polydimethylsiloxane film and serum proteins Finally, Chapter 5 summarizes the conclusions and outlines future research directions.
MORPHOLOGY OF PROTEIN SELF-ASSEMBLY AT THE AIR-
Introduction
The AWI serves as a distinctive tool for guiding proteins, which are biopolymers with uneven distributions of active sites and varying electrostatic and hydrophobic properties, into larger self-assembled structures The unique characteristics of proteins, along with geometric constraints, influence their folding and assembly To effectively assemble complex biological or synthetic systems, promoting interactions in one or two dimensions is essential, with liquid interfaces being particularly advantageous due to their anisotropic forces and two-dimensional confinement Additionally, the self-assembly process is significantly influenced by the chemical and structural complementarity of the building blocks, as well as the interactions of macromolecules like proteins with surrounding solvent molecules.
* This chapter was adapted from Liao, Z.; Lampe, J W.; Ayyaswamy, P S.; Eckmann, D M.; Dmochowski, other solutes 5,6 Thus, solution conditions provide a convenient handle for controlling protein assembly at the AWI
Previous studies using tensiometry, reflectometry, and ellipsometry have investigated the changes in surface excess and structural conformation of proteins at the air-water interface (AWI) under varying solution conditions, including subphase concentration, pH, and ionic strength These techniques provide ensemble-averaged properties of adsorbed proteins on a macroscopic scale To examine assembled structures at the AWI on a nanometer-to-micron scale, imaging methods such as atomic force microscopy (AFM), Brewster angle microscopy (BAM), and fluorescence microscopy have been employed While AFM offers nanometer resolution, it requires transferring interfacial films to a solid surface, risking structural fidelity and temporal resolution BAM, often used in situ with a Langmuir trough, has lower spatial resolution (2 μm) and provides limited kinetic information Notably, earlier research by Gluck et al highlighted fluorescence microscopy's potential as an effective in situ imaging tool for macromolecular self-assembly at the AWI Powers et al further revealed medium-dependent micron-size phase domains of an amphiphilic peptide self-assembled at the AWI, although these findings have yet to be extended to protein systems This study demonstrates, for the first time, the ability to control the microstructure of protein self-assembly at the AWI through systematic variation of solution conditions.
In this chapter, human serum albumin labeled with Texas Red (HSA-TR) serves as a model protein to investigate its self-assembly microstructure through in situ fluorescence microscopy under varying solution conditions HSA is an ideal model due to its well-studied biophysical properties, comprising a single polypeptide chain of 585 amino acids, including 35 cysteines and 59 lysines Notably, 34 cysteines create 17 intramolecular disulfide bridges, while the reactive Cys34 is highly sensitive to redox states X-ray crystallography reveals that HSA exhibits an alpha-helical secondary structure and a heart-shaped tertiary structure, measuring 80 Å on each side and 30 Å in thickness Reflectometry studies indicate that the serum albumin layer at the interface has a thickness of 30–40 Å, with the protein approximated as an ellipsoid, featuring a long axis of 140 Å parallel to the interface and a short axis of 40 Å perpendicular to it.
The study utilized in situ fluorescence microscopy to investigate protein assembly at the air-water interface (AWI) within a small imaging chamber containing approximately 10 µL of solution By covalently attaching a PEG layer to the glass surface, competitive protein adsorption at the liquid-solid interface was minimized, resulting in a stable AWI with negligible evaporation This setup enabled the collection of both static and dynamic data on protein behavior at the AWI with sub-micron spatial resolution over time scales ranging from milliseconds to hours The findings demonstrated the formation of micro-scale protein assemblies at the AWI and underscored the influence of solution conditions, such as ionic strength and reduction potential, on the assembled structures Additionally, kinetic transitions of phase separation at the AWI were observed.
Material and experimental methods
In this study, deionized water (DI water) with a resistivity of 18.2 megohm-cm at 25 °C was utilized for all procedures The phosphate-buffered saline (PBS) solution for fluorescence microscopy was prepared by dissolving NaH2PO4 and NaN3 in DI water, followed by pH adjustment to 7.2 using 1 M HCl, resulting in a final concentration of 10 mM phosphate and 30 mM NaN3 The ionic strength of the PBS was modified by adding NaCl, and Dithiothreitol (DTT) was sourced from Fisher Scientific for protein labeling.
HSA (Sigma-Aldrich) was labeled with the amine-reactive fluorophore Texas Red-X succinimidyl ester (Invitrogen) The labeling process involved dissolving Texas Red-X in dimethylformamide at a concentration of 10 mg/mL, followed by the gradual addition of this concentrated dye solution to an aqueous protein solution containing 2 mg/mL HSA in 0.1 M buffer.
The reaction solution was prepared in a M NaHCO3 buffer at pH 8.3, with a dye to protein molar ratio of 10:1, and stirred for 1 hour at room temperature while covered with aluminum foil Unreacted dyes were removed using a 10-DG column (Bio-Rad), and the labeled proteins were eluted with PBS Further concentration and purification were achieved using 3 kDa cutoff molecular weight centrifugal filter units (Millipore) at 9 krpm for 30 minutes at 4 ºC, followed by dialysis with 10 kDa cassettes (Thermo Scientific) against 1 L of 10 mM phosphate buffer for one week at 4 ºC, also covered with aluminum foil The final concentration of labeled protein was assessed using the Lowry assay (Thermo Scientific) with bovine serum albumin (BSA, Thermo Scientific) as the standard, and the product solution was aliquoted and stored appropriately.
20 ºC for further use c Dye-to-protein ratio (DPR) measurements
DPR was assessed using both UV-Vis spectrometry and MALDI-TOF mass spectrometry In the UV-Vis measurement, labeled proteins were diluted to the correct concentration, and absorbance values were recorded at 280 nm and 595 nm The calculation of DPR was performed using a specific equation.
The absorbance values at 595 nm and 280 nm, denoted as A 595 and A 280, were measured using a diode-array Agilent 89090A spectrophotometer The extinction coefficient (ε) for TR or HSA was provided by the manufacturer, Invitrogen, and supported by literature Additionally, correction factors of 20 and 0.18 were applied to account for the dye's contribution to the absorbance at A 280 Furthermore, MALDI-TOF MS analysis was conducted on HSA and the HSA-TR complex.
MALDI-TOF MS measurements were conducted using a Bruker Daltonic Ultraflex III mass spectrometer, covering a mass range of m/z 10,000-160,000 Prior to analysis, labeled proteins were diluted with HPLC grade water and mixed with a saturated sinapinic matrix solution in a 1:1 ratio After pipetting, the sample droplets were allowed to dry, followed by an on-plate washing step using a 0.1% TFA solution for 10 seconds to remove excess liquid Additionally, circular dichroism (CD) measurements were performed.
CD measurements were performed using a Chirascan CD spectrometer equipped with a Peltier temperature controller HSA and HSA-TR were prepared at a concentration of 0.10 mg/mL in a solution of 10 mM NaCl and 10 mM phosphate buffer at pH 7.2, and then placed in a 0.1 cm pathlength cuvette The Far-UV CD spectra for both HSA and HSA-TR were recorded at 25 ºC across a wavelength range of 190-260 nm, utilizing a scan rate of 0.5 nm/s and a slit bandwidth of 1.0 nm The CD signal was subsequently converted into mean residue ellipticity [θ] according to Equation 2.2.
[θ], in degãcm 2 ãdmol -1 = (millidegrees ì mean residue weight)/(pathlength in millimeters ì concentration in mgãmL -1 ) (Eq 2.2)
Thermal denaturation curves were recorded between 25-95 ºC with a 2 ºC increase and
120 s equilibration time for each step Melting temperature T m was obtained by converting data points in thermal denaturation curves following Eq 2.3, 2.4 and fitting into Eq 2.5 23 using Matlab:
The equation \$\Delta = \Delta N - \Delta D\$ (Eq 2.5) describes the relationship between the ellipticity values of a protein under native conditions, denoted as [θ]N at 25º C, and fully denatured conditions, represented as [θ]D at 95º C The measured ellipticity value at temperature T is indicated as [θ] In this context, K refers to the equilibrium constant, while \$\Delta G(T)\$ represents the Gibbs free energy at temperature T The gas constant is denoted as R, and Tm, the melting temperature, signifies the midpoint of thermal denaturation, with \$\Delta H_m\$ indicating the enthalpy change at T = Tm Additionally, surface pressure measurements are conducted in a Langmuir (LB) trough, followed by film transfer.
Surface pressure was measured with a MicroTroughX Langmuir trough using a Du Noüy-Padday probe A solution of HSA or HSA-TR (0.10 mg/mL) in 10 mM PBS was added to the microtrough, and surface pressure was recorded for one hour following the formation of the air-water interface (AWI) Subsequently, the layer at the AWI was transferred to a freshly cleaved mica surface using the Langmuir-Schaefer technique with a Kibron Microtrough S controller.
A rinsing step using deionized (DI) water was conducted to remove excess salt from the sample surface After rinsing, the sample was allowed to air dry before undergoing characterization through Atomic Force Microscopy (AFM).
The protein layers transferred at the air-water interface (AWI) were analyzed using tapping mode atomic force microscopy (AFM) with Digital Instruments equipment and processed with NanoScope IIIa software (v 5.12) Ultrasharp cantilevers (NSC16, MikroMasch) facilitated the measurements, while WSxM software (Nanotec) was employed to assess the film thickness Additionally, the sample chamber was fabricated and underwent surface modification over a period of 25 hours.
The fluorescence microscope imaging sample chamber was constructed using a thin polydimethylsiloxane (PDMS) layer (1 cm × 1 cm, ~0.5 mm thick) sandwiched between two cover glass pieces The cover glass underwent a cleaning process involving sonication in acetone for 5 minutes, followed by three rinses in deionized (DI) water To prepare the surface –OH groups, the cover glass was sonicated in 200 mM KOH for 20 minutes, then rinsed three times with DI water, sonicated in ethanol for 5 minutes, and allowed to air dry.
PDMS is prepared using a commercial kit (Sylgard 184, Dow Corning) by mixing the elastomer and curing agent in a 10:1 weight ratio After thorough mixing and degassing, the liquid PDMS is poured into a Petri dish and cured at 80 °C for 2.5 hours Once cured, the PDMS slab is peeled off, cut into 1 cm × 1 cm pieces, and a 0.7-cm diameter cylindrical hole is punched in the center The PDMS pieces are then washed with deionized water and dried with compressed air before being placed on a cleaned cover glass Immediate bonding occurs between the PDMS and the cover glass, creating a chamber that serves as a sample holder for protein solutions, effectively preventing leaks through conformal contact.
The bottom cover glass of the inner chamber was treated with mPEG-silane to minimize protein adsorption, while the inner PDMS surfaces were oxidized and similarly modified using a solution phase method The mPEG-silane was stored at -20°C and brought to room temperature before being dissolved in a 5% acetic acid/methanol solution at a concentration of 250 mg/mL This solution was added to the chamber using a glass pipet, and a second cover glass was placed on top to prevent evaporation The assembly was incubated at 40°C for 30 minutes, after which the top cover glass was removed, and the chamber was rinsed with deionized water, dried with argon, and stored at -20°C until imaging with confocal and epi-fluorescence techniques.
Confocal images were obtained with an Olympus IX81 inverted microscope, utilizing a hyperspectral CCD camera for epi-fluorescence imaging The 40X water immersion objective lens excited the sample dyes using a 543 nm laser or a mercury lamp with a 530-550 nm excitation filter, while emission was collected in the 575-655 nm range Image analysis was performed using ImageJ software.
Results and discussion
a Characterization of HSA-TR in comparison with HSA
Fluorescent probes are commonly utilized to investigate protein behaviors; however, there are concerns regarding the impact of exogenous fluorophores on protein conformation This is particularly critical in surface-related studies, where it is essential to thoroughly characterize dye-labeled proteins in comparison to their native counterparts, as the dye molecules can modify both the conformation and hydrophobicity of the proteins.
In our research, we aimed for minimal labeling by optimizing the dye and protein concentrations in the labeling reaction, and we characterized the dye-protein ratio (DPR) using UV-Vis spectroscopy and MALDI-TOF MS The DPR values obtained were 1.3 from UV-Vis spectroscopy and 1.5 from MALDI-TOF MS, as illustrated in Figure 2.1 The proteins were protonated by the matrix, with peaks corresponding to A + 2H\(^+\), A + H\(^+\), and 2A + H\(^+\) for HSA and HSA-TR, where "A" denotes the protein molecule.
H + peaks of HSA and HSA-TR the mass difference (△m) = m(HSA-TR) - m(HSA) 1119 g/mol Because MW(TR) = 723 g/mol, thus DPR = △m/MW(TR) = 1.5
The comparison of secondary structures between HSA and HSA-TR was conducted using circular dichroism (CD) spectroscopy, revealing that the far-UV CD spectra of both proteins closely overlap, suggesting that the Texas Red (TR) dye does not significantly alter the characteristic α-helical structure of HSA Additionally, thermal denaturation curves indicate that HSA-TR exhibits greater thermal stability, with a melting temperature 5 ºC higher than that of HSA This stability enhancement may be attributed to the interaction of the Texas Red dye with ligand binding sites in HSA, as supported by existing literature.
The surface pressure curves of HSA-TR and HSA were analyzed to evaluate their surface activities As shown in Figure 2.3, HSA exhibits a marginally higher surface pressure than HSA-TR, with a difference of less than 1 mN/m, which falls within the experimental error range Therefore, the impact of Texas Red on the surface activity of HSA is minimal, if it exists at all.
Using Atomic Force Microscopy (AFM), we examined the HSA-TR or HSA layer formed at the Air-Water Interface (AWI) under similar conditions (C = 0.1 mg/mL at neutral pH) as reported in previous studies on BSA The morphology of the transferred films displayed interconnected small spherical dots, resembling the structure of BSA documented in the literature Analysis of the AFM images indicated that the thickness of the protein layer is approximately 3 nm, consistent with the dimensions of a flat-lying HSA molecule as determined by neutron reflectivity studies.
Thus, by CD, tensiometry and AFM, we conclude that minimally labeled, near-native HSA provides a useful model protein sample for studying assembly at the AWI by fluorescence microscopy
Figure 2.1 MALDI-MS spectra of HSA and HSA-TR
The black line represents the spectra of HSA, while the red line indicates the spectra of HSA-TR The peaks, from left to right, correspond to A + 2H\(^+\), A + H\(^+\), and 2A + H\(^+\) for HSA and HSA-TR, with "A" denoting the protein molecule.
Figure 2.2 Characterization of HSA and HSA-TR by CD spectroscopy
The far-UV circular dichroism (CD) spectra of human serum albumin (HSA) and its variant HSA-TR were analyzed at 25 °C, revealing distinct differences The thermal denaturation curves indicated a melting temperature (T m) of 71 °C for HSA and 76 °C for HSA-TR These curves were obtained by recording data between 25 °C and 95 °C, with a 2 °C increment and a 120-second equilibration time for each step.
Figure 2.3 Surface pressure of HSA and HSA-TR
C protein = 0.10 mg/mL in 10 mM PBS buffer, pH = 7.2 Each curve is the average of two independent experiments
Figure 2.4 AFM images of HSA and HSA-TR films formed at the AWI and transferred onto mica surface
Initial subphase concentrations were 0.10 mg/mL, pH = 7.2 Top row: AFM images of (A) HSA, (B) HSA-
TR Scale bar: 1.0 àm Bottom row: height of protein films plotted along yellow dashed line drawn in (A, black) and (B, red) b Effect of surface modification of imaging chamber with PEG
The imaging chamber, a microliter-sized reservoir for protein solutions, experiences competition between protein adsorption on solid surfaces and at the air-water interface (AWI) To mitigate this issue, we employ mPEG-silane to covalently modify the exposed hydroxyl groups on the inner surfaces of the chamber, creating a layer of PEG chains that effectively reduces protein adsorption, as illustrated in Figure 2.5.
Confocal microscopy was used to monitor the efficiency of HSA-TR adsorption reduction Before PEG surface modification, a small peak of fluorescence intensity from HSA-TR was detected at the solid-liquid interface (Figure 2.6 A2) However, post-modification, confocal imaging revealed no surface excess at the interface (Figure 2.6 B2) Similar results were observed on the PEG-modified PDMS surface, as illustrated in Figures 2.6 A3 and B3.
Figure 2.5 Covalent modification of surface hydroxyl groups by mPEG-silane
Figure 2.6 Confocal fluorescence images of HSA-TR in sample chamber before and after surface modification
XZ-plane images of the HSA-TR solution (C(HSA-TR) = 0.10 mg/mL in PBS buffer) were captured in both unmodified (A1) and modified (B1) sample chambers The intensity plot along the z-axis for A1 and B1 is shown in (A2) and (B2) Additionally, XY-plane images were focused on the edge of the unmodified (A3) and modified (B3) chambers with a concentration of C(HSA-TR) = 0.50 mg/mL in PBS buffer This study utilizes CLSM to image protein assembly at the air-water interface (AWI).
In a specially designed sealed chamber, HSA-TR was imaged with a protein aqueous phase of 10 μL and a thickness of 100-200 μm, coexisting with a similar layer of air The air-water interface (AWI) was aligned parallel to the XY plane of the microscope stage, allowing for the collection of images either at the interface (XY plane images) or by scanning perpendicularly (XZ plane images) with a step size of 0.1 μm HSA-TR, dissolved in PBS with an ionic strength of 193 mM, exhibited a uniform fluorescent layer at concentrations of 0.050 mg/mL or higher, as observed through confocal microscopy In contrast, at concentrations of 0.025 mg/mL or lower, a highly heterogeneous structure was detected at the interface, revealing HSA-TR assembled into micrometer-sized fractal structures XZ-plane images confirmed these findings, showing a uniform fluorescent layer at higher concentrations and a discontinuous layer at lower concentrations, suggesting a surface-saturating bulk-solution concentration (C_B sat).
) which fell between 0.025 mg/mL and 0.050 mg/mL under the condition of ionic strength at 193 mM in PBS Below CB sat
Adsorbed proteins do not completely cover the air-water interface (AWI), leading to the development of protein domains at the interface Previous studies utilizing tensiometry and ellipsometry have determined that the critical concentration of bovine serum albumin (BSA) required to effectively cover the AWI ranges from 0.01 to 0.1 mg/mL.
Although researchers in the field of protein adsorption generally accept the concept of
, 32,33 very few studies have revealed the microstructure of assembled proteins at concentrations below CB sat
A study by Lee et al indicated that mechanical heterogeneity exists in a β-lactoglobulin layer at the air-water interface (AWI) even at low concentrations Early two-dimensional lattice-based simulations predicted that fractal networks could form through diffusion-limited aggregation under similar conditions The XY-plane images at the AWI confirm the presence of fractal assembly and a heterogeneous protein layer at low subphase concentrations.
Figure 2.7 XY and XZ plane fluorescent images of protein assembly at the AWI
The study presents a sample chamber and inverted microscope setup to capture confocal images of HSA-TR in PBS at pH 7.2 and an ionic strength of 193 mM The XY-plane images illustrate HSA-TR concentrations of 0.050 mg/mL and 0.025 mg/mL, with images utilizing 4X optical zoom for enhanced detail Additionally, XZ-plane images provide insights into the protein assembly influenced by ionic strength and reducing agents, with a scale bar indicating 20 µm.
We investigated the impact of varying ionic strength and redox state on protein assemblies at the air-water interface (AWI) After introducing the solutions into the chamber, we allowed the interfaces to age for one hour before capturing images A concentration of 0.010 mg/mL human serum albumin (HSA) was used in the experiments.
Conclusions
In conclusion, we utilized fluorescence microscopy as an in situ technique to investigate protein behavior at the air-water interface (AWI) over timescales ranging from milliseconds to hours Our findings reveal that below surface-saturating bulk-solution concentrations, the self-assembly of the model protein HSA-TR exhibits diverse morphologies in response to variations in solution conditions such as protein concentration, ionic strength, and redox state Our method effectively detects both the static and dynamic behavior of HSA-TR at the AWI and is readily applicable for studying other macromolecular self-assembly processes Additionally, this approach can be extended to examine the glass-water interface, offering potential for monitoring competitive adsorption of surface-active species at gas-liquid and solid-liquid interfaces This study highlights the potential for controlling protein assembly at the AWI for various biomaterials applications.
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SEMI-QUANTITATIVE CONFOCAL LASER SCANNING
Material and experimental setup
Phosphate buffered saline (PBS) was utilized as the buffer in this chapter, prepared by dissolving 136 mM NaCl, 8.1 mM Na2HPO4, 2 mM KCl, 1.5 mM KH2PO4, and 31 mM NaN3 in deionized water, and maintained at pH 7.4 at room temperature Human fibrinogen labeled with Oregon Green (HFib-OG, Cat# F7496) was sourced from Life Technologies, while human serum albumin (HSA, Cat# A3782) was obtained from Sigma and used without further purification Proteins were dissolved in PBS through vortexing and filtered using sterilized Millex® Syringe Filter Units (Cat# SLGV004SL, Millipore) with a 0.22 μm pore size A 10% solution of Pluronic F-127 (MW~12500, Cat# P-6866) was also purchased from Life Technologies HSA was covalently labeled with fluorophores as described in Chapter 2 IIb, and the concentrations of protein or dye-labeled protein stock solutions were determined using the Lowry assay (Thermo Scientific).
The CLSM setup utilized in this study mirrors the configuration outlined in Chapter 2, employing an argon laser (488 nm, 30 mW) to excite Oregon Green dye and a HeNe laser (543 nm, 1 mW) for Texas Red Fluorescence recovery after photobleaching (FRAP) experiments were conducted on the same confocal microscope equipped with an Olympus SIM scanner unit, which was used to photobleach small regions of the protein layer at the AWI for 20 seconds Confocal images of these regions were captured before, during, and after the photobleaching process using a 543 nm laser with a dwell time of 2 μs per pixel.
III Results and discussion a Methodology
We developed a semi-quantitative method using a droplet system, where a 10 μL droplet was placed on a coverslip in a MatTek Petri dish, with the lid closed to minimize evaporation To control humidity, a donut-shaped sponge soaked in DI water surrounded the coverslip area The Petri dish was cleaned with HPLC grade ethanol and water after each use, and the focal plane was set approximately 150 μm above the bottom coverglass to reduce light scattering A fluorescence image of an HSA-TR droplet revealed a bright rim at the air-water interface (AWI), indicating HSA-TR concentration due to adsorption A rectangular region of interest (ROI) was defined, measuring 200 pixels in length and 50 pixels in width, and fluorescence intensity was averaged along the y-axis and plotted along the x-axis.
We investigated the competition between HFib-OG and F-127 at the air-water interface (AWI) by analyzing intensity profiles Our results, illustrated in Figure 3.2, show that HFib-OG adsorbs at the interface, as indicated by the intensity peaks at both concentrations in the absence of F-127 However, when F-127 was introduced at a concentration of 80 μM, these intensity peaks diminished around 7 μm from the origin of the region of interest (ROI), suggesting that F-127 effectively competes with HFib-OG for adsorption at the interface This significant finding prompted us to enhance the confocal laser scanning microscopy (CLSM) method to further explore the competitive interactions between F-127 and plasma proteins at the AWI Utilizing the imaging setup detailed in Chapter 2, we examined both the adsorption competition of the protein/F-127 mixture and the phase behavior of the interfacial layer.
The semi-quantitative method was enhanced using a custom imaging chamber system, allowing for the study of AWI through both XY scans for morphological data and XZ scans for semi-quantitative intensity data Details on the chamber's fabrication can be found in Chapter 2 An XZ plane image, shown in Figure 3.3 A, represents a 90-degree clockwise rotation of the ROI depicted in Figure 3.1 B Image analysis was conducted using MATLAB, facilitating the examination of multiple frames, with all original codes available in Appendix I Images were imported into MATLAB via the “readtif” program, and subsequent calculations were performed using the “PC” program, which converted TIFF format RGB images to grayscale and subtracted background intensity from the ROI for intensity analysis The intensity data was then plotted along the z-axis, as illustrated in Figure 3.3 B.
The total intensity I total in ROI is determined to be:
I j is the averaged intensity of pixel j along x-axis, n is the total number of pixels along z-axis of ROI Then average bulk intensity I avgbulk is:
In this study, we selected a starting pixel, denoted as \( m \), from the bulk region located at the right shoulder of the interface intensity peak Subsequently, we calculated the bulk intensity \( I_{\text{bulk}} \) and the interface intensity \( I_{\text{int}} \) using specific formulas.
(Eq 3.4) z is the first pixel at the left shoulder of the interface intensity peak where I z ≥ I avgbulk
Finally, partition coefficient (P) which represents the partitioning of dye-labeled protein between bulk phase and interface phase was determined: int avgbulk
The definition of P is similar to that of surface excess, with surface excess being a two-dimensional quantity and P being unitless If we assume that the bulk concentration changes little after protein adsorption at the interface, the surface concentration can be approximately related to P multiplied by Cbulk.
Cbulk cannot precisely quantify the surface concentration are discussed in the last section of this chapter
Figure 3.1 Experimental setup and data analysis of the droplet system to study protein adsorption at the AWI
The article presents a cartoon illustrating the imaging setup, with a horizontal dashed line marking the image slice location It features a representative image of the AWI of a BSA-TR aqueous droplet, highlighting a yellow box that denotes the region of interest (ROI) for intensity analysis, with a scale bar of 20 μm Additionally, the intensity profile of the ROI is provided, showing the averaged pixel intensity along the y-axis plotted against the x-axis.
Figure 3.2 Intensity profiles of F-127 and HFib-OG mixtures show that F-127 blocks protein adsorption
Plot shows four intensity profiles measured at 1 h near the droplet edge
Figure 3.3 Analysis of an XZ plane image to obtain partition coefficient
An XZ plane image at a depth of 100μm was obtained while scanning the air-water interface (AWI) of a HSA-TR sample in the imaging chamber The partition coefficient was calculated from the intensity profile, where interfacial fluorescence corresponds to the total intensity of the orange area assigned to a single pixel, and bulk fluorescence represents the average pixel intensity of the green area The partition coefficient is defined as the ratio of interfacial fluorescence (indicated by the orange dashed line) to the average bulk fluorescence (represented by the green dashed line) Additionally, the study examines the adsorption competition between HSA-TR and F-127 at the AWI.
In our study, we utilized HSA-TR due to its status as the most abundant protein in human serum and the well-characterized surface activity and protein conformation before and after labeling We measured the partition coefficient of HSA-TR at varying bulk concentrations to establish a standard curve, observing a significant drop in P from 0.05 mg/mL to 0.25 mg/mL, followed by a gradual decrease above 0.5 mg/mL This behavior aligns with the notion that the energy barrier for protein adsorption at the interface increases with higher surface coverage Once the interface is fully covered by protein, the proteins tend to adsorb loosely in the sub-layer, resulting in minimal changes in P, as supported by previous microscopy studies.
Here is the rewritten paragraph:Notably, our previous findings in Chapter 2 revealed that a uniform layer was formed at the air-water interface (AWI) at a concentration of 0.05 mg/mL, as observed by Confocal Laser Scanning Microscopy (CLSM) However, a closer examination of the partition coefficient trend suggests that a densely packed interfacial layer of HSA-TR only forms at a significantly higher concentration of approximately 0.5 mg/mL.
We investigated the partition coefficient of the HSA-TR/F-127 mixture by fixing the HSA-TR concentration at 0.1 mg/mL and 0.5 mg/mL while varying the F-127 concentration After one hour, we measured the partition coefficient (P) and found that adding F-127 from 0 to 0.01 mg/mL slightly decreased P compared to HSA-TR-only samples As the F-127 concentration increased further, phase separation occurred, leading to an averaged P value from multiple areas At higher concentrations, fluorescence intensity at the interface diminished, causing P to drop to 0, indicating that HSA-TR was completely displaced from the interface Notably, a higher concentration of HSA-TR necessitated a greater concentration of F-127 to induce lateral phase separation and displace the proteins from the interface.
The morphology of the interfacial layer varied with surfactant concentration and time After 10 minutes of AWI formation, the interfacial structures transitioned from a uniformly fluorescent layer at low surfactant concentrations to a layer exhibiting "cracks." As the concentration of F-127 increased, the dark areas in the interfacial layer expanded, while the fluorescent regions diminished into small "islands." At even higher concentrations of F-127, the strong fluorescent signal from the interface was entirely lost, indicating that the initial interfacial layers were heterogeneous, formed by the premixed solution with a specific protein/surfactant ratio.
After an extended aging period of the interface, the fluorescent signal in the bottom row decreased compared to the top, as shown in Figure 3.6 This blurring of the boundary between bright and dark regions suggests improved mixing due to the diffusion of protein and F-127 within the film In the case of HSA-TR at a concentration of 0.50 mg/mL mixed with the F-127 solution, the ratio of n(HSA-TR) to n(F-127) indicates significant changes in the mixture's properties.
127) in which phase separation was observed at 10 min after interface formation was 6-2
Figure 3.4 Partition coefficient of HSA-TR as a function of bulk concentration
Figure 3.5 Change of partition coefficient P with increase in F-127 concentration
Red dashed lines represent the P of HSA-TR at identical bulk concentrations in the absence of F-127 The shaded areas highlight the concentration range where phase separations were detected at the AWI Measurements of P were taken one hour after the solution was introduced into the chamber.
Figure 3.6 Phase separation in adsorbed HSA/F-127 layer at the AWI