Exploiting the Fc base of IgG antibodies to create functional nanoparticle conjugates.

The structures of the Fc base of various IgG antibodies have been examined with a view to understanding how this region can be used to conjugate IgG to nanoparticles. The base structure is found to be largely consistent across a range of species and subtypes, comprising a hydrophobic region surrounded by hydrophilic residues, some of which are charged at physiological conditions. In addition, atomistic Molecular Dynamics simulations were performed to explore how model nanoparticles interact with the base using neutral and negatively charged gold nanoparticles. Both types of nanoparticle interacted readily with the base, leading to an adaptation of the antibody base surface to enhance the interactions. Furthermore, these interactions left the rest of the domain at the base of the Fc region structurally intact. This implies that coupling nanoparticles to the base of an IgG molecule is both feasible and desirable, since it leaves the antibody free to interact with its surroundings so that antigen-binding functionality can be retained. These results will therefore help guide future attempts to develop new nanotechnologies that exploit the unique properties of both antibodies and nanoparticles.

Estimating Binding Energies of π-Stacked Aromatic Dimers Using Force Field-Driven Molecular Dynamics.

π-π stacking are omnipresent interactions, crucial in many areas of chemistry, and often studied using quantum chemical methods. Here, we report a simple and computationally efficient method of estimating the binding energies of stacked polycyclic aromatic hydrocarbons based on steered molecular dynamics. This method leverages the force field parameters for accurate calculation. The presented results show good agreement with those obtained through DFT at the ωB97X-D3/cc-pVQZ level of theory. It is demonstrated that this force field-driven SMD method can be applied to other aromatic molecules, allowing insight into the complexity of the stacking interactions and, more importantly, reporting π-π stacking energy values with reasonable precision.

Nanoparticle Metrology of Silicates Using Time-Resolved Multiplexed Dye Fluorescence Anisotropy, Small Angle X-ray Scattering, and Molecular Dynamics Simulations.

We investigate the nanometrology of sub-nanometre particle sizes in industrially manufactured sodium silicate liquors at high pH using time-resolved fluorescence anisotropy. Rather than the previous approach of using a single dye label, we investigate and quantify the advantages and limitations of multiplexing two fluorescent dye labels. Rotational times of the non-binding rhodamine B and adsorbing rhodamine 6G dyes are used to independently determine the medium microviscosity and the silicate particle radius, respectively. The anisotropy measurements were performed on the range of samples prepared by diluting the stock solution of silicate to concentrations ranging between 0.2 M and 2 M of NaOH and on the stock solution at different temperatures. Additionally, it was shown that the particle size can also be measured using a single excitation wavelength when both dyes are present in the sample. The recovered average particle size has an upper limit of 7.0 ± 1.2 Å. The obtained results were further verified using small-angle X-ray scattering, with the recovered particle size equal to 6.50 ± 0.08 Å. To disclose the impact of the dye label on the measured complex size, we further investigated the adsorption state of rhodamine 6G on silica nanoparticles using molecular dynamics simulations, which showed that the size contribution is strongly impacted by the size of the nanoparticle of interest. In the case of the higher radius of curvature (less curved) of larger particles, the size contribution of the dye label is below 10%, while in the case of smaller and more curved particles, the contribution increases significantly, which also suggests that the particles of interest might not be perfectly spherical.

Impact of the Crystal Structure of Silica Nanoparticles on Rhodamine 6G Adsorption: A Molecular Dynamics Study.

Understanding the mechanism of adsorption of Rhodamine 6G (R6G) to various crystal structures of silica nanoparticles (SNPs) is important to elucidate the impact of dye size when measuring the size of the dye-SNP complex via the time-resolved fluorescence anisotropy method. In this work, molecular dynamics (MD) simulations were used to get an insight into the R6G adsorption process, which cannot be observed using experimental methods. It was found that at low pH, α-Cristobalite structured SNPs have a strong affinity to R6G; however, at high pH, more surface silanol groups undergo ionization when compared with α-Quartz, preventing the adsorption. Therefore, α-Quartz structured SNPs are more suitable for R6G adsorption at high pH than the α-Cristobalite ones. Furthermore, it was found that stable adsorption can occur only when the R6G xanthene core is oriented flat with respect to the SNP surface, indicating that the dye size does not contribute significantly to the measured size of the dye-SNP complex. The requirement of correct dipole moment orientation indicates that only one R6G molecule can adsorb on any sized SNP, and the R6G layer formation on SNP is not possible. Moreover, the dimerization process of R6G and its competition with the adsorption has been explored. It has been shown that the highest stable R6G aggregate is a dimer, and in this form, R6G does not adsorb to SNPs. Finally, using steered molecular dynamics (SMD) with constant-velocity pulling, the binding energies of R6G dimers and R6G complexes with both α-Quartz and α-Cristobalite SNPs of 40 Å diameter were estimated. These confirm that R6G adsorption is most stable on 40 Å α-Quartz at pH 7, although dimerization is equally possible.

Chitin and Chitosan Binding to the α-Chitin Crystal: A Molecular Dynamics Study.

Understanding the binding of chitosan oligomers to the surface of a chitin nanocrystal is important for improving the enzymatic deacetylation of chitin and for the design of chitin/chitosan composite films. Here, we study the binding of several chito-oligomers to the (100) surface of an α-chitin crystal using molecular dynamics (MD), steered MD, and umbrella sampling. The convergence of the free energy was carefully considered and yielded a binding energies of -12.5 and -2 kcal mol-1 for 6-monomer-long chitin and uncharged chitosan oligomers, respectively. We also found that the results for the umbrella sampling were consistent with the force profile from the steered MD and with classical MD simulations of the adsorption process. Our results give insight into the molecular-scale interactions, which can be helpful for the design of new chitin composite films. Furthermore, the free energy curves we present can be used to validate coarse-grained models for chitin and chitosan, which are necessary to study the self-assembly of chitin crystals due to the long time scale of the process.

Functionalisation of Inorganic Material Surfaces with Staphylococcus Protein A: A Molecular Dynamics Study.

Staphylococcus protein A (SpA) is found in the cell wall of Staphylococcus aureus bacteria. Its ability to bind to the constant Fc regions of antibodies means it is useful for antibody extraction, and further integration with inorganic materials can lead to the development of diagnostics and therapeutics. We have investigated the adsorption of SpA on inorganic surface models such as experimentally relevant negatively charged silica, as well as positively charged and neutral surfaces, by use of fully atomistic molecular dynamics simulations. We have found that SpA, which is itself negatively charged at pH7, is able to adsorb on all our surface models. However, adsorption on charged surfaces is more specific in terms of protein orientation compared to a neutral Au (111) surface, while the protein structure is generally well maintained in all cases. The results indicate that SpA adsorption is optimal on the siloxide-rich silica surface, which is negative at pH7 since this keeps the Fc binding regions free to interact with other species in solution. Due to the dominant role of electrostatics, the results are transferable to other inorganic materials and pave the way for new diagnostic and therapeutic designs where SpA might be used to conjugate antibodies to nanoparticles.

Multiple myeloma: therapeutic delivery of antibodies and aptamers.

Multiple myeloma is the second most common hematological malignancy in adults, accounting for 2% of all cancer-related deaths in the UK. Current chemotherapy-based regimes are insufficient, as most patients relapse and develop therapy resistance. This review focuses on current novel antibody- and aptamer-based therapies aiming to overcome current therapy limitations, as well as their respective limitations and areas of improvement. The use of computer modeling methods, as a tool to study and improve ligand-receptor alignments for the use of novel therapy development will also be discussed, as it has become a rapid, reliable and comparatively inexpensive method of investigation.

Simulating Peptide Monolayer Formation: GnRH-I on Silica.

Molecular dynamics (MD) simulations can provide a detailed view of molecule behaviour at an atomic level, which can be useful when attempting to interpret experiments or design new systems. The decapeptide gonadotrophin-releasing hormone I (GnRH-I) is known to control fertility in mammals for both sexes. It was previously shown that inoculation with silica nanoparticles (SiNPs) coated with GnRH-I makes an effective anti-fertility vaccine due to how the peptide adsorbs to the nanoparticle and is presented to the immune system. In this paper, we develop and employ a protocol to simulate the development of a GnRH-I peptide adlayer by allowing peptides to diffuse and adsorb in a staged series of trajectories. The peptides start the simulation in an immobile state in solution above the model silica surface, and are then released sequentially. This facile approach allows the adlayer to develop in a natural manner and appears to be quite versatile. We find that the GnRH-I adlayer tends to be sparse, with electrostatics dominating the interactions. The peptides are collapsed to the surface and are seemingly free to interact with additional solutes, supporting the interpretations of the GNRH-I/SiNP vaccine system.

Direct Immobilization of Engineered Nanobodies on Gold Sensors.

Single-domain antibodies, known as nanobodies, have great potential as biorecognition elements for sensors because of their small size, affinity, specificity, and robustness. However, facile and efficient methods of nanobody immobilization are sought that retain their maximum functionality. Herein, we describe the direct immobilization of nanobodies on gold sensors by exploiting a modified cysteine strategically positioned at the C-terminal end of the nanobody. The experimental data based on secondary ion mass spectrometry, circular dichroism, and surface plasmon resonance, taken together with a detailed computational work (molecular dynamics simulations), support the formation of stable and well-oriented nanobody monolayers. Furthermore, the nanobody structure and activity is preserved, wherein the nanobody is immobilized at a high density (approximately 1 nanobody per 13 nm2). The strategy for the spontaneous nanobody self-assembly is simple and effective and possesses exceptional potential to be used in numerous sensing platforms, ranging from clinical diagnosis to environmental monitoring.

Structural Analysis of Anti-Hapten Antibodies to Identify Long-Range Structural Movements Induced by Hapten Binding.

Antibodies are well known for their high specificity that has enabled them to be of significant use in both therapeutic and diagnostic applications. Antibodies can recognize different antigens, including proteins, carbohydrates, peptides, nucleic acids, lipids, and small molecular weight haptens that are abundantly available as hormones, pharmaceuticals, and pesticides. Here we focus on a structural analysis of hapten-antibody couples and identify potential structural movements originating from the hapten binding by comparison with unbound antibody, utilizing 40 crystal structures from the Protein Data Bank. Our analysis reveals three binding surface trends; S1 where a pocket forms to accommodate the hapten, S2 where a pocket is removed when the hapten binds, and S3 where no pockets changes are found. S1 and S2 are expected for induced-fit binding, whereas S3 indicates that a pre-existing population of optimal binding antibody conformation exists. The structural analysis reveals four classifications of structural reorganization, some of which correlate to S2 but not to the other binding surface changes. These observations demonstrate the complexity of the antibody-antigen interaction, where structural changes can be restricted to the binding sites, or extend through the constant domains to propagate structural changes. This highlights the importance of structural analysis to ensure successful and compatible transformation of small antibody fragments at the early discovery stage into full antibodies during the subsequent development stages, where long-range structural changes are required for an Fc effector response.

Biomolecular interactions with nanoparticles: applications for coronavirus disease 2019.

Nanoparticles are small particles sized 1-100 nm, which have a large surface-to-volume ratio, allowing efficient adsorption of drugs, proteins, and other chemical compounds. Consequently, functionalized nanoparticles have potential diagnostic and therapeutic applications. A variety of nanoparticles have been studied, including those constructed from inorganic materials, biopolymers, and lipids. In this review, we focus on recent work targeting the severe acute respiratory syndrome coronavirus 2 virus that causes coronavirus disease (COVID-19). Understanding the interactions between coronavirus-specific proteins (such as the spike protein and its host cell receptor angiotensin-converting enzyme 2) with different nanoparticles paves the way to the development of new therapeutics and diagnostics that are urgently needed for the fight against COVID-19, and indeed for related future viral threats that may emerge.

Antibody-protein binding and conformational changes: identifying allosteric signalling pathways to engineer a better effector response.

Numerous monoclonal antibodies have been developed successfully for the treatment of various diseases. Nevertheless, the development of biotherapeutic antibodies is complex, expensive, and time-consuming, and to facilitate this process, careful structural analysis beyond the antibody binding site is required to develop a more efficacious antibody. In this work, we focused on protein antigens, since they induce the largest antibody changes, and provide interesting cases to compare and contrast. The structures of 15 anti-protein antibodies were analysed to compare the antigen-bound/unbound forms. Surprisingly, three different classes of binding-induced changes were identified. In class (B1), the antigen binding fragment distorted significantly, and we found changes in the loop region of the heavy chain's constant domain; this corresponds well with expected allosteric movements. In class (B2), we found changes in the same loop region without the overall distortion. In class (B3), these changes did not present, and only local changes at the complementarity determining regions were found. Consequently, structural analysis of antibodies is crucial for therapeutic development. Careful evaluation of allosteric movements must be undertaken to develop better effector responses, especially during the transformation of these antibodies from small fragments at the discovery stage to full antibodies at the subsequent development stages.

Critical role of tyrosine-20 in formation of gold nanoclusters within lysozyme: a molecular dynamics study.

Lysozyme is one of the most commonly used proteins for encapsulating gold nanoclusters, yielding Ly-AuNC complexes. While possible applications of Ly-AuNCs in environmental, biological and trace metal sensing in solution have been demonstrated, there is currently a poor understanding of the physical characteristics of the Ly-AuNC complex. In this study we have employed fully atomistic molecular dynamics simulations to gain an understanding of the formation of Au clusters within the protein. It was found that in order to form AuNCs in the simulations, an approach of targeted insertion of Au atoms at a critical surface residue was needed. Tyrosine is known to be crucial for the reduction of Au salts experimentally, and our simulations showed that Tyr20 is the key residue for the formation of an AuNC beneath the protein surface in the α-helical domain. It is hoped these observations will aid future improvements and modification of Ly-AuNCs via alterations of the alpha-helix domain or Tyr20.

Rationalising drug delivery using nanoparticles: a combined simulation and immunology study of GnRH adsorbed to silica nanoparticles.

Silica nanoparticles (SiNPs) have been shown to have significant potential for drug delivery and as adjuvants for vaccines. We have simulated the adsorption of GnRH-I (gonadotrophin releasing hormone I) and a cysteine-tagged modification (cys-GnRH-I) to model silica surfaces, as well as its conjugation to the widely-used carrier protein bovine serum albumin (BSA). Our subsequent immunological studies revealed no significant antibody production was caused by the peptide-SiNP systems, indicating that the treatment was not effective. However, the testosterone response with the native peptide-SiNPs indicated a drug effect not found with cys-GnRH-I-SiNPs; this behaviour is explained by the specific orientation of the peptides at the silica surface found in the simulations. With the BSA systems, we found significant testosterone reduction, particularly for the BSA-native conjugates, and an antibody response that was notably higher with the SiNPs acting as an adjuvant; this behaviour again correlates well with the epitope presentation predicted by the simulations. The range of immunological and hormone response can therefore be interpreted and understood by the simulation results and the presentation of the peptides to solution, paving the way for the future rational design of drug delivery and vaccine systems guided by biomolecular simulation.

Adsorption of Fibronectin Fragment on Surfaces Using Fully Atomistic Molecular Dynamics Simulations.

The effect of surface chemistry on the adsorption characteristics of a fibronectin fragment (FNIII8⁻10) was investigated using fully atomistic molecular dynamics simulations. Model surfaces were constructed to replicate self-assembled monolayers terminated with methyl, hydroxyl, amine, and carboxyl moieties. It was found that adsorption of FNIII8⁻10 on charged surfaces is rapid, specific, and driven by electrostatic interactions, and that the anchoring residues are either polar uncharged or of opposing charge to that of the targeted surfaces. On charged surfaces the presence of a strongly bound layer of water molecules and ions hinders FNIII8⁻10 adsorption. In contrast, adsorption kinetics on uncharged surfaces are slow and non-specific, as they are driven by van der Waals interactions, and the anchoring residues are polar uncharged. Due to existence of a positively charged area around its cell-binding region, FNIII8⁻10 is available for subsequent cell binding when adsorbed on a positively charged surface, but not when adsorbed on a negatively charged surface. On uncharged surfaces, the availability of the fibronectin fragment's cell-binding region is not clearly distinguished because adsorption is much less specific.

Energy Landscape of Negatively Charged BSA Adsorbed on a Negatively Charged Silica Surface.

We study the energy landscape of the negatively charged protein bovine serum albumin adsorbed on a negatively charged silica surface at pH 7. We use fully atomistic molecular dynamics (MD) and steered MD (SMD) to probe the energy of adsorption and the pathway for the surface diffusion of the protein and its associated activation energy. We find an adsorption energy ∼1.2 eV, which implies that adsorption is irreversible even on experimental time scales of hours. In contrast, the activation energy for surface diffusion is ∼0.4 eV so that it is observable on the MD simulation time scale of 100 ns. This analysis paves the way for a more detailed understanding of how a protein layer forms on biomaterial surfaces, even when the protein and surface share the same electrical polarity.

Probing beta amyloid aggregation using fluorescence anisotropy: experiments and simulation.

The aggregation of beta amyloid (Ab) protein is associated with the development of Alzheimer's disease. In this work we monitor Ab aggregation using fluorescence anisotropy, a technique that provides information on the rotational diffusion of the fluorescing tyrosine (Tyr) side chains. We also perform Monte Carlo (MC) and fully atomistic Molecular Dynamics (MD) simulations to interpret the experiments. The experimental results show that there are two different rotational timescales contributing to the anisotropy. Our MC simulation captures this behaviour in a coarse-scale manner, and, more importantly, shows that the Tyr side chains must have their movements restricted in order to reproduce the anisotropy. The MD simulations provide a molecular scale view, and indeed show that aggregation restricts the Try side chains to yield anisotropy in line with the experimental results. This combination of experiment and simulation therefore provides a unique insight into the aggregation process, and we suggest how this approach might be used to gain further information on aggregating protein systems.

Tyrosine Rotamer States in Beta Amyloid: Signatures of Aggregation and Fibrillation.

During the early stages of ß amyloid (Ab) peptide aggregation, toxic oligomers form which have been recognized as a likely cause of Alzheimer's disease. In this work, we use fully atomistic molecular dynamics simulation to study the amorphous aggregation of the peptide as well as model ß-sheet protofibril structures. In particular, we study the rotamer states of the single fluorescent tyrosine (Tyr) residue present in each Ab. We find that the occupation of the four previously identified rotamers is different for monomeric and amorphous aggregates because of the differing environments of the Tyr side-chains. Surprisingly, we also identify two new rotamers that uniquely appear for the ß-sheet structures, so that together the rotamers provide distinct signatures for the different stages of aggregation and fibrillation. We propose that these rotamers could be identified in fluorescence spectroscopy, with each rotamer having a distinct fluorescence lifetime because of its different exposures to the solvent. The identification of the two new rotamers therefore provides a new means to probe amyloid formation kinetics and to monitor the effect of additives including prospective drugs.

Thinking outside the Laboratory: Analyses of Antibody Structure and Dynamics within Different Solvent Environments in Molecular Dynamics (MD) Simulations.

Monoclonal antibodies (mAbs) have revolutionized the biomedical field, directly influencing therapeutics and diagnostics in the biopharmaceutical industry, while continuing advances in computational efficiency have enabled molecular dynamics (MD) simulations to provide atomistic insight into the structure and function of mAbs. Despite the success of MD tools, further optimizations are still required to enhance the computational efficiency of complex mAb simulations. This issue can be tackled by changing the way the solvent system is modelled to reduce the number of atoms to be tracked but must be done without compromising the accuracy of the simulations. In this work, the structure of the IgG2a antibody was analyzed in three solvent systems: explicit water and ions, implicit water and ions, and implicit water and explicit ions. Root-mean-square distance (RMSD), root-mean-square fluctuations (RMSF), and interchain angles were used to quantify structural changes. The explicit system provides the most atomistic detail but is ~6 times slower in its exploration of configurational space and required ~4 times more computational time on our supercomputer than the implicit simulations. Overall, the behavior of the implicit and explicit simulations is quantifiably similar, with the inclusion of explicit ions in the implicit simulation stabilizing the antibody to reproduce well the statistical fluctuations of the fully explicit system. Therefore, this approach holds promise to maximize the use of computational resources to explore antibody behavior.

Bovine Serum Albumin Adsorption at a Silica Surface Explored by Simulation and Experiment.

Molecular details of BSA adsorption on a silica surface are revealed by fully atomistic molecular dynamics (MD) simulations (with a 0.5 µs trajectory), supported by dynamic light scattering (DLS), zeta potential, multiparametric surface plasmon resonance (MP-SPR), and contact angle experiments. The experimental and theoretical methods complement one another and lead to a wider understanding of the mechanism of BSA adsorption across a range of pH 3-9. The MD results show how the negatively charged BSA at pH7 adsorbs to the negatively charged silica surface, and reveal a unique orientation with preserved secondary and tertiary structure. The experiments then show that the protein forms complete monolayers at ∼ pH6, just above the protein's isoelectric point (pH5.1). The surface contact angle is maximum when it is completely coated with protein, and the hydrophobicity of the surface is understood in terms of the simulated protein conformation. The adsorption behavior at higher pH > 6 is also consistently interpreted using the MD picture; both the contact angle and the adsorbed protein mass density decrease with increasing pH, in line with the increasing magnitude of negative charge on both the protein and the surface. At lower pH < 5 the protein starts to unfold, and the adsorbed mass dramatically decreases. The comprehensive picture that emerges for the formation of oriented protein films with preserved native conformation will help guide efforts to create functional films for new technologies.