Protein In Bulk Solution Research Articles

PyMBE: The Python-based molecule builder for ESPResSo.

We present the Python-based Molecule Builder for ESPResSo (pyMBE), an open source software application to design custom coarse-grained (CG) models, as well as pre-defined models of polyelectrolytes, peptides, and globular proteins in the Extensible Simulation Package for Research on Soft Matter (ESPResSo). The Python interface of ESPResSo offers a flexible framework, capable of building custom CG models from scratch. As a downside, building CG models from scratch is prone to mistakes, especially for newcomers in the field of CG modeling, or for molecules with complex architectures. The pyMBE module builds CG models in ESPResSo using a hierarchical bottom-up approach, providing a robust tool to automate the setup of CG models and helping new users prevent common mistakes. ESPResSo features the constant pH (cpH) and grand-reaction (G-RxMC) methods, which have been designed to study chemical reaction equilibria in macromolecular systems with many reactive species. However, setting up these methods for systems, which contain several types of reactive groups, is an error-prone task, especially for beginners. The pyMBE module enables the automatic setup of cpH and G-RxMC simulations in ESPResSo, lowering the barrier for newcomers and opening the door to investigate complex systems not studied with these methods yet. To demonstrate some of the applications of pyMBE, we showcase several case studies where we successfully reproduce previously published simulations of charge-regulating peptides and globular proteins in bulk solution and weak polyelectrolytes in dialysis. The pyMBE module is publicly available as a GitHub repository (https://github.com/pyMBE-dev/pyMBE), which includes its source code and various sample and test scripts, including the ones that we used to generate the data presented in this article.

Relationship between aggregation of therapeutic proteins and agitation parameters: Acceleration and frequency

An increase in protein aggregates during transportation should be suppressed in therapeutic protein products because the aggregates have a potential risk of immunogenicity. In this study, three protein solutions in vials were exposed to tri-axial vibration with various combinations of frequency and acceleration using a transportation test system to investigate the relationship between low g-force stresses and protein aggregate generation. The number concentration of micron aggregates detected by flow imaging analysis increased markedly when the acceleration and frequency of agitation were within a specific range, in other words, above a threshold. This threshold was common among the three protein solutions. The suppression of micron aggregate formation by adding a surfactant suggested that agitation above the threshold increased micron aggregates mainly via interface-mediated routes. Notably, agitation, including agitation below the threshold, accelerated spontaneous oligomerization (nanometer aggregate generation) of proteins in bulk solution even in the presence of the surfactant. Studies of stability against mechanical stresses (e.g., a random vibration test to simulate actual shipment, with a time-compressed setting by increasing acceleration) need to be performed and discussed with careful consideration of the threshold for generating micron aggregates.

The effect of charged residue substitutions on the thermodynamics of protein-surface interactions.

The interactions of proteins with surfaces are important in both biological processes and biotechnologies. In contrast to decades of study regarding the biophysics of proteins in bulk solution, however, our mechanistic understanding of the biophysics of proteins interacting with surfaces remains largely qualitative. In response, we have set to explore quantitatively the thermodynamics of protein-surface interactions. In this work, we explore systematically the role of electrostatics in modulating the interaction between proteins and charged surfaces. In particular, we use electrochemistry to explore the extent to which a macroscopic, hydroxyl-coated surface held at a slightly negative potential affects the folding thermodynamics of surface-attached protein variants with different composition of charged amino acids. Doing so, we find that attachment to the surface generally leads to a net stabilization, presumably due to excluded volume effects that reduce the entropy of the unfolded state. The magnitude of this stabilization, however, is strongly correlated with the charged-residue content of the protein. In particular, we find statistically significant correlations with both the net charge of the protein, with greater negative charge leading to less stabilization by the surface, and with the number of arginines, with more arginines leading to greater stabilization. Such findings refine our understanding of protein-surface interactions, providing in turn a guiding rationale to achieve the functional deposition of proteins on artificial surfaces for implementation in, for example, protein-based biotechnologies.

Coarse-grained simulation of the translational and rotational diffusion of globular proteins by dissipative particle dynamics.

With simplified interactions and degrees of freedom, coarse-grained (CG) simulations have been successfully applied to study the translational and rotational diffusion of proteins in solution. However, in order to reach larger lengths and longer timescales, many CG simulations employ an oversimplified model for proteins or an implicit-solvent model in which the hydrodynamic interactions are ignored, and thus, the real kinetics are more or less unfaithful. In this work, we develop a CG model based on the dissipative particle dynamics (DPD) that can be universally applied to different types of proteins. The proteins are modeled as a group of rigid DPD beads without conformational changes. The fluids (including solvent and ions) are also modeled as DPD beads. The electrostatic interactions between charged species are explicitly considered by including charge distributions on DPD particles. Moreover, a surface friction between the protein and fluid beads is applied to control the slip boundary condition. With this model, we investigate the self-diffusion of a single globular protein in bulk solution. The translational and rotational diffusion coefficients of the protein can be tuned by the surface frictional constant to fit the predictions of the Stokes-Einstein (SE) relation. We find that both translational and rotational diffusion coefficients that meet with the prediction of the SE relation based on experimental results of the hydrodynamic radius are reached at almost the same frictional constant for different types of proteins. Such scaling behavior indicates that the model can be applied to simulate the translational and rotational diffusion together for various types of proteins.

Insight into Heterogeneous Distribution of Protein Aggregates at the Surface Layer Using Attenuated Total Reflection-Fourier Transform Infrared Spectroscopic Imaging.

Monoclonal antibodies (mAbs) have been used as therapeutics for the last few decades. It is necessary to investigate the stability of these mAbs under stress conditions and to elucidate aggregation mechanisms as a means of developing approaches which minimize the problem. Attenuated total reflection (ATR)-FTIR spectroscopic imaging allows probing of a sample at a depth of penetration of around 0.5-5 μm, which makes it suitable for the study of aggregated proteins when accumulated as a layer close to the surface of the ZnSe internal reflection element (IRE). Here, macro ATR-FTIR spectroscopic imaging, along with a variable angle of incidence accessory, have been used to differentiate between the secondary structure of proteins in bulk solution and those that have precipitated onto or near the ZnSe IRE surface. IgG spectra obtained from protein samples in individual wells have been averaged, extracted, and preprocessed, and the Amide I bands of the protein samples were compared and further analyzed to reveal protein distribution at the ZnSe IRE surface. These findings show depth profiling of IgG aggregates at the ZnSe IRE surface (0.5-5 μm) and do not follow a trend of decreasing protein presence with an increasing angle of incidence or increasing depth of penetration, suggesting an irregular distribution of aggregates in the z-direction.

Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution

The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed. The time of GroES-induced dissociation of a denatured protein from the GroEL surface was estimated using the stopped-flow technique and found to be much shorter than the proposed time of the GroEL ATPase cycle.

Understanding the Biophysics of Protein-Surface Interactions

Despite the biological and biotechnological importance of protein-surface interactions, our understanding of how and why they occur is still limited: What are the thermodynamic consequences of the interaction of proteins with surfaces? Why do proteins generally remain folded and functional on biological surfaces and cell membranes, but often unfold, adhere to, and inactivate on artificial surfaces? To respond to these questions we need to understand the biophysical origins of protein-surface interactions, however the absence of experimental methods to measure the thermodynamics of such interactions has so far precluded quantitative analysis. In response, we have developed an approach to measure the free energy of protein-surface interactions, which we have employed to explore the extent to which attachment to a specific, macroscopic surface alters the thermodynamic stability of protein L. We have achieved so by modifying protein L with the redox reporter methylene blue and then covalently tethering its N-terminus to a gold electrode passivated with a hydroxyl-terminated alkanethiol monolayer. Denaturant-induced unfolding of the surface-attached protein alters the ease with which the methylene blue reporter transfers electrons to the surface of the electrode, and therefore electrochemical techniques, such as square-wave voltammetry, allow monitoring the unfolding to extract protein stability. Comparing the thus obtained stability of the surface-attached protein to that of the same protein in bulk solution we find that surface-attachment stabilizes the protein due to excluded volume effects that restrict the conformational entropy of the unfolded state. We have also explored the role of macromolecular crowding, solvent composition and electrostatics to find that their surface biophysics are markedly different from those in bulk solution. We believe that our studies refine our understanding of the biophysics underlying protein-surface interactions, which may in turn improve the design of protein-surface pairs for protein-incorporating biotechnologies such as protein-based sensors.

Temperature-Induced Denaturation of BSA Protein Molecules for Improved Surface Passivation Coatings.

Bovine serum albumin (BSA) is the most widely used protein for surface passivation applications, although it has relatively weak, nonsticky interactions with hydrophilic surfaces such as silica-based materials. Herein, we report a simple and versatile method to increase the stickiness of BSA protein molecules adsorbing onto silica surfaces, resulting in up to a 10-fold improvement in blocking efficiency against serum biofouling. Circular dichroism spectroscopy, dynamic light scattering, and nanoparticle tracking analysis showed that temperature-induced denaturation of BSA proteins in bulk solution resulted in irreversible unfolding and protein oligomerization, thereby converting weakly adhesive protein monomers into a more adhesive oligomeric form. The heat-treated, denatured BSA oligomers remained stable after cooling. Room-temperature quartz crystal microbalance-dissipation and localized surface plasmon resonance experiments revealed that denatured BSA oligomers adsorbed more quickly and in larger mass quantities onto silica surfaces than native BSA monomers. We also determined that the larger surface contact area of denatured BSA oligomers is an important factor contributing to their more adhesive character. Importantly, denatured BSA oligomers were a superior passivating agent to inhibit biofouling on silica surfaces and also improved Western blot application performance. Taken together, the findings demonstrate how temperature-induced denaturation of BSA protein molecules can lead to improved protein-based coatings for surface passivation applications.

Do Not Drop: Mechanical Shock in Vials Causes Cavitation, Protein Aggregation, and Particle Formation

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 μs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond time course. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size-exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues.

Reverse Micelles As a Platform for Dynamic Nuclear Polarization in Solution NMR of Proteins

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ∼−93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules.

Formation of Dielectric Layers and Charge Regulation in Protein Adsorption at Biomimetic Interfaces

Protein charge is an important parameter in the understanding of protein interactions and function. Proteins are subject to dynamic charge regulation, that is, the influence of the local environment (such as charged interfaces and biopolymers) on protein charge. Charge regulation is governed by differences in the dielectric and electrostatic environment between adsorbed protein and the free protein in bulk solution. In this work protein charge regulation is addressed experimentally by employing electrochemistry at interfaces between two immiscible electrolyte solutions (ITIES) as well as theoretically by developing a new protein adsorption model at ITIES. Electrochemistry at ITIES is shown to be particularly well suited to study protein charge regulation as the adsorbed protein experiences a different dielectric environment compared to the bulk phase and the external control of the water/oil potential difference allows systematic studies on how potential induced ion gradients affect protein charge. The theoretical model incorporates all the features of the experimental system and specifically takes into account protein charge regulation at ITIES as well as the impact of the formation of dielectric layers on the experimentally observed impedance. The model parameters include the protein charge-pH profile, bulk pH, and the overall potential difference. It is shown that the formation of a dielectric layer and the associated charge regulation are the main factors dictating the observed experimental behavior. Finally, the theoretical model is used to interpret literature results, and the consistency between the model and the relatively large data set suggests that the model may be used more generally for understanding and predicting protein adsorption.

Protein Conformation and Supercharging with DMSO from Aqueous Solution

The efficacy of dimethyl sulfoxide (DMSO) as a supercharging reagent for protein ions formed by electrospray ionization from aqueous solution and the mechanism for supercharging were investigated. Addition of small amounts of DMSO to aqueous solutions containing hen egg white lysozyme or equine myoglobin results in a lowering of charge, whereas a significant increase in charge occurs at higher concentrations. Results from both near-UV circular dichroism spectroscopy and solution-phase hydrogen/deuterium exchange mass spectrometry indicate that DMSO causes a compaction of the native structure of these proteins at low concentration, but significant unfolding occurs at ~63% and ~43% DMSO for lysozyme and myoglobin, respectively. The DMSO concentrations required to denature these two proteins in bulk solution are ~3–5 times higher than the concentrations required for the onset of supercharging, consistent with a significantly increased concentration of this high boiling point supercharging reagent in the ESI droplet as preferential evaporation of water occurs. DMSO is slightly more basic than m-nitrobenzyl alcohol and sulfolane, two other supercharging reagents, based on calculated proton affinity and gas-phase basicity values both at the B3LYP and MP2 levels of theory, and all three of these supercharging reagents are significantly more basic than water. These results provide additional evidence that the origin of supercharging from aqueous solution is the result of chemical and/or thermal denaturation that occurs in the ESI droplet as the concentration of these supercharging reagents increases, and that proton transfer reactivity does not play a significant role in the charge enhancement observed.

Investigation of Electrophoretic Exclusion Method for the Concentration and Differentiation of Proteins

This work presents a technique termed as "electrophoretic exclusion" that is capable of differentiation and concentration of proteins in bulk solution. In this method, a hydrodynamic flow is countered by the electrophoretic velocity to prevent a species from entering into a channel. The separation can be controlled by changing the flow rate or applied electric potential in order to exclude a certain species selectively while allowing others to pass through the capillary. The exclusion of various proteins is investigated using a flow-injection regime of the method. Concentration of myoglobin of up to 1200 times the background concentration in 60 s was demonstrated. Additionally, negatively charged myoglobin was separated from a solution containing negatively charged allophycocyanin. Cationic cytochrome c was also differentiated from a solution with allophycocyanin. The ability to differentially transport species in bulk solution enables parallel and serial separation modes not available with other separations schemes.

Enabling Site-Resolved Measurement of Hydration Water-Protein Interactions by Solution NMR

The interaction between a protein and solvating water is fundamental to its stability, dynamics, and function. In principle, solution NMR methods can be employed to identify and characterize the location and dynamics of specific solvent-protein interactions. Unfortunately, the residence times of water on the surface of a protein in bulk solution are such that the elegant experiments of Wa1/4thrich and coworkers are defeated. Here, we take advantage of the slowed dynamics of water under nanoconfinement and tap the full potential of these experiments to obtain the first site-resolved, solution NMR measurements of hydration water-protein interactions. Reverse micellar encapsulation slows water dynamics enough to allow detectable build-up of direct protein-water dipolar magnetization exchange while at the same time slowing hydrogen exchange kinetics by up to two orders of magnitude, thereby reducing indirect contributions to the spectra. Using amide hydrogens as a probe, we are able to detect literally dozens of hydration waters on the surface of encapsulated ubiquitin. Three-dimensional 15N-NOESY and ROESY spectra of ubiquitin in reverse micelles have been obtained. The relative intensities of NOE/ROE cross peaks at the water resonance confirms that a minority of the detected hydration waters have long residence times (ωτ > 1), while the majority of detected solvation waters have a range of shorter residence times. This approach appears to open the door to global site-resolved descriptions of protein hydration dynamics and therefore represent a major step forward in the quest to understand the motional links between proteins and solvent. Supported by NSF-0842814 and NIH F32GM074376 (M.P.) and F32GM08709 (N.N.).

Separation of residual fatty acids from aqueous solutions using an agitated solution of protein and membrane filtration

Wastewaters from food processing plants often contain residual fatty acids in solubilized form. They are often difficult to separate by using ultrafiltration membranes due to their small size (∼1 nm) and tendency to condense in membrane pores causing severe flux reduction. These difficulties can be overcome by binding fatty acids to large protein molecules so that the resulting complex can be easily retained on a membrane. Binding and filtration may be carried out in a single equipment such as an agitated column. The rate of mass transfer of the fatty acid molecules to the surface of protein molecules influences efficiency of separation and utilization of the protein in a continuous process. The mass transfer coefficient during filtration of fatty acid solutions through agitated protein solutions was studied using caprylate and bovine serum albumin as model fatty acid–protein system. An agitated cell fitted with a fully retentive membrane (30 kDa nominal molecular weight cutoff) to the protein was used at 100 kPa. Fatty acid in the permeate was monitored by periodic analysis of permeate samples for organic carbon. A linear driving force model was used to evaluate the mass transfer coefficients. At pH 4.9 and stirring rate at 500 rpm, the mass transfer coefficient remains around 6 min −1 until approximately 60% of saturation of BSA, and then it rapidly drops. At low agitation (50 rpm) mass transfer coefficient gradually increased to 7.5 min −1 before a gradual decline, indicating involvement of other fatty acid retention mechanisms in addition to binding to protein in bulk solution. Under low agitation and pH, increased protein fouling and condensation of fatty acid in fouled layers of the protein could have resulted in apparent increase of the mass transfer coefficient and may provide a potentially effective process to remove large amounts of these residual fatty acids.

Structure and dynamics of egg white ovalbumin adsorbed at the air/water interface

The molecular properties of egg white ovalbumin adsorbed at the air/water interface were studied using infrared reflection absorption spectroscopy (IRRAS) and time-resolved fluorescence anisotropy (TRFA) techniques. Ovalbumin adsorbed at the air/water interface adopts a characteristic partially unfolded conformation in which the content of the beta-sheet is 10% lower compared to that of the protein in bulk solution. Adsorption to the interface leads to considerable changes in the rotational dynamics of ovalbumin. The results indicate that the end-over-end mobility of the ellipsoidal protein becomes substantially restricted. This is likely to reflect a preferential orientation of the protein at the interface. Continuous compression of surface layers of ovalbumin causes local aggregation of the protein, resulting in protein-network formation at the interface. The altered protein-protein interactions contribute to the strong increase in surface pressure observed.

Membrane Proteins in Bulk Solution Can Be Used for Quasi-Elastic Neutron Scattering Studies: The Case for the Photochemical Reaction Center

The potential to measure the internal dynamics of a membrane protein, the bacterial photochemical reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides, was investigated by incoherent quasi-elastic neutron scattering (IQENS) and neutron spin−echo spectroscopy. To isolate and maximize the scattering intensity attributed to the protein, selective deuteration was employed. This deuteration was achieved by embedding detergent-purified RC proteins in a solution containing perdeuterated D28-n-octyl-β-d-glucopyranoside, a sugar−hydroxy detergent. Therefore, the IQENS signal essentially arises only from the motions, which are resolved in time, of the nonexchangeable hydrogen atoms of the protein. This work has established that with a suitable biochemical protocol detailed measurements on the internal dynamics of any integral membrane protein can be undertaken.

II. The photochemistry of proteins

The examination of monolayers has been found to afford a quantitative method of investigation of the photochemical behaviour of many organic compounds of high molecular weight and appears to be especially suitable for macromolecular substances of physiological interest such as the proteins (Rideal 1932; Mitchell 1936 a , 1937 a ; Rideal and Mitchell 1937). The photochemical reactions occuring in the compounds spread in monolayers light, are observed by measurement of the changes in phase boundary potential and surface pressure; the light intensity is measured by chemical reference to proteins and related similar compounds, and, as a preliminary investing, the photochemical properties of the keto-imino linkage have been examined. The present work on the photochemistry of monolayers of typical proteins was undertaken from the point of view of radiation therapy, with the object of providing elementary data for interpretation of the biological actions of radiation. The investigation is quantitative and is accordingly limited to proteins for which the amino acid analyses are sufficiently complete, namely, wheat gliadin, zein, insulin and ovalbumin. The application of the analytic data to the interpration of the results of the photochemical experiments demands a detailed knowledge of the physical processes of light absorption in monolayers and of the photochemical significance of the absorption regard to the molecular configuration of the monolayers are necessary. The results of photochemical experiments on protein in bulk solution are required in the discussion of the chemical interpretation of the physical measurements on the monolayers.