Advanced computational approaches to understand protein aggregation.

Protein aggregation is a widespread phenomenon implicated in debilitating diseases like Alzheimer's, Parkinson's, and cataracts, presenting complex hurdles for the field of molecular biology. In this review, we explore the evolving realm of computational methods and bioinformatics tools that have revolutionized our comprehension of protein aggregation. Beginning with a discussion of the multifaceted challenges associated with understanding this process and emphasizing the critical need for precise predictive tools, we highlight how computational techniques have become indispensable for understanding protein aggregation. We focus on molecular simulations, notably molecular dynamics (MD) simulations, spanning from atomistic to coarse-grained levels, which have emerged as pivotal tools in unraveling the complex dynamics governing protein aggregation in diseases such as cataracts, Alzheimer's, and Parkinson's. MD simulations provide microscopic insights into protein interactions and the subtleties of aggregation pathways, with advanced techniques like replica exchange molecular dynamics, Metadynamics (MetaD), and umbrella sampling enhancing our understanding by probing intricate energy landscapes and transition states. We delve into specific applications of MD simulations, elucidating the chaperone mechanism underlying cataract formation using Markov state modeling and the intricate pathways and interactions driving the toxic aggregate formation in Alzheimer's and Parkinson's disease. Transitioning we highlight how computational techniques, including bioinformatics, sequence analysis, structural data, machine learning algorithms, and artificial intelligence have become indispensable for predicting protein aggregation propensity and locating aggregation-prone regions within protein sequences. Throughout our exploration, we underscore the symbiotic relationship between computational approaches and empirical data, which has paved the way for potential therapeutic strategies against protein aggregation-related diseases. In conclusion, this review offers a comprehensive overview of advanced computational methodologies and bioinformatics tools that have catalyzed breakthroughs in unraveling the molecular basis of protein aggregation, with significant implications for clinical interventions, standing at the intersection of computational biology and experimental research.

Tuning Electrostatic Interactions To Control Orientation of GFP Protein Adsorption on Silica Surface.

The adsorption of green fluorescent protein (GFP) on silica surfaces has been the subject of growing interest due to its potential applications in various fields, including biotechnology and biomedicine. In this study, we used all-atom molecular dynamics simulations to investigate the charge-driven adsorption of wild type GFP and its supercharged variants on silica surfaces. The results showed that the positively charged variant of GFP adsorbed on the negatively charged silica surface with minimal loss in its secondary structure. Further studies were conducted to understand the role of surface charge distribution on two other positively charged variants of GFP, and the results showed that the orientation of GFP on silica can be easily tuned by careful mutations of the charged amino acid residues on the GFP. This study provides valuable molecular insights into the role of electrostatic-driven adsorption of GFP and highlights the importance of charge interactions in the adsorption process.

Molecular Insights into the Inhibitory Role of α-Crystallin against γD-Crystallin Aggregation.

Cataracts, a major cause of global blindness, contribute significantly to the overall prevalence of blindness. The opacification of the lens, resulting in cataract formation, primarily occurs due to the aggregation of crystallin proteins within the eye lens. Despite the high concentration of these crystallins, they remarkably maintain the lens transparency and refractive index. α-Crystallins (α-crys), acting as chaperones, play a crucial role in preventing crystallin aggregation, although the exact molecular mechanism remains uncertain. In this study, we employed a combination of molecular docking, all-atom molecular dynamics simulations, and advanced free energy calculations to investigate the interaction between γD-crystallin (γD-crys), a major structural protein of the eye lens, and α-crystallin proteins. Our findings demonstrate that α-crys exhibits an enhanced affinity for the NTD2 and CTD4 regions of γD-crys. The NTD2 and CTD4 regions form the interface between the N-terminal domain (NTD) and the C-terminal domain (CTD) of the γD-crys protein. By binding to the interface region between the NTD and CTD of the protein, α-crys effectively inhibits the formation of domain-swapped aggregates and mitigates protein aggregation. Analysis of the Markov state models using molecular dynamics trajectories confirms that minimum free energy conformations correspond to the binding of the α-crystallin domain (ACD) of α-crys to NTD2 and CTD4 that form the interdomain interface.

Effect of mutations on the folding and stability of γD-crystallin protein.

Interprotein interactions between the partially unfolded states of γD-crystallin (γD-crys) protein are known to cause cataracts. Therefore, understanding the unfolding pathways of native γD-crys is extremely crucial to delineate their aggregation mechanism. In this study, we have performed extensive all-atom Molecular Dynamics simulations with explicit solvent to understand the role of the critical residues that drive the stability of the motifs and domains of γD-crys in its wild type and mutant forms. Our findings show that while the individual motifs of wild type are not stable in the native form, the individual domains remain structurally stable at 425K. This enhanced stability of the domain was attributed to the hydrophobic interactions between the motifs. Single and double point mutations of the domains with negatively charged aspartic and glutamic acid amino acid residues (I3E, W42D, W42E, I3D/W42D, I3E/W42E, and L92D/W157D) decreases the structural stability, leading to unfolding of individual domains of γD-crys. We believe that our study sheds light on the weakest links of γD-crys, along with the role of interactions stabilizing the domains. Further, this study bolsters and provides a better understanding of the domain swapping mechanism of aggregation of γD-crys.Communicated by Ramaswamy H. Sarma.

Hydrophobicity─A Single Parameter for the Accurate Prediction of Disordered Regions in Proteins.

The prediction of disordered regions in proteins is crucial for understanding their functions, dynamics, and interactions. Intrinsically disordered proteins (IDPs) play a key role in many biological processes like cell signaling, recognition, and regulation, but experimentally determining these regions can be challenging due to their high mobility. To address this challenge, we present an algorithm called HydroDisPred (HDP). HDP uses a single parameter, the fraction of hydrophobicity (λ) in each segment of the protein, to accurately predict disordered regions. The algorithm was validated using experimental data from the DisProt database and was found to be on par and, in some cases, more effective than the existing algorithms. HDP is a simple and effective method for identifying disordered regions in proteins, and its prediction is not affected by the availability of training data, unlike other ML approaches. The application is housed in the web server and can be accessed through the URL https://proseqanalyser.iitgn.ac.in/hydrodispred/.

Understanding the helical stability of charged peptides.

Cationic helical peptides play a crucial role in applications such as anti-microbial and anticancer activity. The activity of these peptides directly correlates with their helicity. In this study, we have performed extensive all-atom molecular dynamics simulations of 25 Lysine-Leucine co-polypeptide sequences of varying charge density ( λ ) and patterns. Our findings showed that, an increase in the charge density on the peptide leads to a gradual decrease in the helicity up to a critical charge density λ c . Beyond λ c , a complete helix to coil transition was observed. The decrease in the helicity is correlated with the increased number of water molecules in first solvation shell, solvent-exposed surface area, and a higher value of the radius of gyration of the peptide.

Understanding asymmetry effects at low grafting density on the self-assembly of polyion grafted nanoparticles.

Grafting of spherical nanoparticles (NPs) with polymeric ligands has been an effective way of controlling the dispersion state of NPs either in the matrix or in solution. Despite the fast evolving synthesis techniques, it is still experimentally difficult to precisely control the position of tethers on the surface of NPs. At low grafting density, due to the surface anisotropy, a wide range of assemblies could be achieved depending on the position of the tethers on the NP surface. This may pose a challenge in interpreting data from scattering and electron micrographs. In the current manuscript, we use coarse-grained molecular dynamics simulations to study the effect of graft angles (positions) on the self-assembly of NPs grafted with oppositely charged polyions at low grafting density driven by coulombic interactions. Our study shows that the NPs grafted with oppositely charged polyions self-assemble into a wide range of morphologies like dimers, rings, strings, coils, tetramers and aggregates depending on the angle between the grafts. Furthermore, the morphologies obtained in the case of semi-flexible and rigid-chains as tethers are more sensitive to the graft positions (angles) compared to that of flexible chains. Molecular dynamics studies carried out at low grafting densities of the system comprising an equimolar mixture of nanoparticles grafted with polyions at varying grafting angles showed that the polyions grafted at low/high grafting angles preferentially interacted with oppositely charged polyion grafts tethered at low/high graft angles.

Understanding the stimuli responsive behavior of polyion grafted nanoparticles in the presence of salt and polyelectrolytes.

The design of nanoparticles (NPs) that respond to external stimuli like pH, temperature, and electric or magnetic fields has found immense interest in various fields of nanotechnology like nanomedicine, drug delivery, and cancer therapy. Nanoparticles grafted with polymeric ligands have been extensively used as building blocks in the directed self assembly of nanoparticles. These moieties not only assemble into various morphologies but also respond to a wide range of external stimuli. In this work, we have used coarse grained molecular dynamics simulations to understand the stimuli-responsive behavior of assemblies of NPs grafted with oppositely charged polyions (PGNs) in the presence of salt and polyelectrolytes. At low grafting density, a transformation from ring morphology to form dimers/strings/dispersed NPs was observed upon addition of divalent/trivalent salts. NPs grafted with longer grafts showed higher stability to remain as rings compared to shorter grafts. The change in NP morphology was a direct consequence of preferential interaction of the polyaion grafts with the oppositely charged salt ions compared to the oppositely charged grafts on the NPs. At fixed salt valency, the size of the salt ion, concentration and molecular connectivity played a crucial role in the stimuli responsive behavior of polyion grafted NPs in solutions. Further, in the presence of polyelectrolytes, these transitions occurred at lower monomer valency due to the stronger electrostatic interactions between the grafted chains and oppositely charged free polyelectrolytes in solutions. Disordered and ordered aggregates assemblies formed at higher grafting density were broken into smaller NP assemblies in the presence of salt. Drug encapsulation studies in the presence of salt and polyelectrolytes were performed on model drug moieties in order to demonstrate the potential use of the modelled stimuli responsive nanoparticle assemblies in drug delivery applications.

Controlled 3D assembly and stimuli responsive behavior of DNA and peptide functionalized gold nanoparticles in solutions.

DNA mediated directed self assembly of gold nanoparticles (AuNPs) has garnered immense interest due to its ability to precisely control supramolecular assemblies. Most experimental works have relied on utilizing the complementary interactions between the DNA strands to drive the self assembly of AuNPs grafted with DNA strands. In the present work, we have leveraged DNA-peptide interactions to tune the self assembly and stimuli responsive behavior of AuNPs grafted with single stranded DNA (ssDNA) and poly-L-lysine (PLL) chains. Our findings show that the electrostatic interactions between the negatively charged ssDNA grafts and positively charged PLL grafts, drive the self assembly of AuNPs of different sizes into 3D nanostructures. The transmission electron micrographs confirm that the smaller AuNPs grafted with PLL chains form a corona around the large AuNPs grafted with ssDNA like the petals around a flowery core to drive aggregation of large AuNPs. When the grafting of ssDNA and PLL on the different sized AuNPs is swapped, aggregates of large AuNPs mediated by the ssDNA grafts on the smaller AuNPs were observed. The presence of excess ssDNA/PLL chains in solutions affected both the morphology and the mechanism of aggregate formation. Coarse-grain molecular dynamics simulations qualitatively match the experimental findings and provided a scientific rationale to the above findings highlighting the role of chain entropy, molecular connectivity, and charge correlations on the self assembly of AuNPs.

Charge-Driven Self-Assembly of Polyelectrolyte-Grafted Nanoparticles in Solutions.

Nanoparticle self-assembly in solution has gained immense interest due to the enhanced optical, chemical, magnetic, and electrical properties which manifest at the macroscale. Material properties in bulk are a direct consequence of the morphology of these nanoparticles in solutions. Precise control on the orientation, spatial arrangement, shape, size, composition, and control over the interactions of individual nanoparticles play a key role in enhancing their properties. While previous studies have used asymmetry in the nanoparticle and/or the use of linker grafts, nanoparticles grafted with polyelectrolyte grafts provide us a wide parameter space to control and tune their self-assembly in solutions. In this study, we have performed coarse-grained molecular dynamics simulations to understand the charge-driven self-assembly of spherical nanoparticles grafted with polyelectrolyte chains. Nanoparticles grafted with either positively or negatively charged polyelectrolyte chains self-assemble to different structures driven by both excluded volume and electrostatic interactions. Our study shows that by tuning the graft density, the chain length, and the charge density of the grafts, we could build and control a variety of self-assembled structures ranging from rings, dimers, strings, coil-like aggregates, and disordered-to-ordered aggregates.

Understanding the role of hydrophobic patches in protein disaggregation.

Protein folding is a very complex process and, so far, the mechanism of folding still intrigues the research community. Despite a large conformational space available (O(1047) for a 100 amino acid residue), most proteins fold into their native state within a very short time. While small proteins fold relatively fast (a few microseconds) large globular proteins may take as long as several milliseconds to fold. During the folding process, the protein synthesized in the ribosome is exposed to the crowded environment of the cell and is easily prone to misfolding and aggregation due to interactions with other proteins or biomacromolecules present within the cell. These large proteins, therefore, rely on chaperones for their folding and repair. Chaperones are known to have hydrophobic patchy domains that play a crucial role in shielding the protein against misfolding and disaggregation of aggregated proteins. In the current article, Monte Carlo simulations carried out in the framework of the hydrophobic-polar (H-P) lattice model indicate that hydrophobic patchy domains drastically reduce the inter-protein interactions and are efficient in disaggregating proteins. The effectiveness of the disaggregation depends on the size and distribution of these patches on the surface and also on the strength of the interaction between the protein and the surface. Further, our results indicate that when the patch is complementary to the exposed hydrophobic patch of the protein, protein disaggregation is accompanied by stabilization of the protein even relative to its bulk behavior due to favorable protein-surface interactions. We believe that these findings shed light on the role of the class of chaperones known as heat shock proteins (Hsps) on protein disaggregation and refolding.

Mitochondrial Impairment by Cyanine-Based Small Molecules Induces Apoptosis in Cancer Cells.

Mitochondrion, the powerhouse of the cells, has emerged as one of the unorthodox targets in anticancer therapy due to its involvement in several cellular functions. However, the development of small molecules for selective mitochondrial damage in cancer cells remained limited and less explored. To address this, in our work, we have synthesized a natural product inspired cyanine-based 3-methoxy pyrrole small molecule library by a concise strategy. This strategy involves Vilsmeier and Pd(0) catalyzed Suzuki cross-coupling reactions as key steps. The screening of the library members in HeLa cervical cancer cells revealed two new molecules that localized into subcellular mitochondria and damaged them. These small molecules perturbed antiapoptotic (Bcl-2/Bcl-xl) and pro-apoptotic (Bax) proteins to produce reactive oxygen species (ROS). Molecular docking studies showed that both molecules bind more tightly with the BH3 domain of Bcl-2 proteins compared to obatoclax (a pan-Bcl-2 inhibitor). These novel small molecules arrested the cell cycle in the G0/G1 phase, cleaved caspase-3/9, and finally prompted late apoptosis. This small molecule-mediated mitochondrial damage induced remarkably high cervical cancer cell death. These unique small molecules can be further explored as chemical biology tools and next-generation organelle-targeted anticancer therapy.

Surface Patterning for Enhanced Protein Stability: Insights from Molecular Simulations.

Reduced activity of enzymes upon immobilization is a major challenge for the industrial use of enzymes. Enzyme-surface interactions and interactions between the immobilized enzymes are thought of as primary reasons for the reduced activity. In the current paper, we study the thermal and structural stability of proteins on a patterned hydrophobic surface in the framework of a hydrophobic-polar lattice model. Our results indicate that, while a homogeneous hydrophobic surface denatures the proteins, carefully patterned surfaces can dramatically increase the stability of adsorbed proteins. The size, shape, and the distance between surface patterns play a significant role in determining the stability of proteins. When the spacing between the patterns is large, maximum stability is observed when the surface pattern is complementary to the exposed hydrophobic domain of the protein, while at smaller spacing, patterns with lower hydrophobicity stabilize the protein more compared to the complementary pattern. The findings from the paper can be rationalized to design novel enzyme-specific surfaces for immobilization with enhanced enzymatic activity.

Role of confinement, molecular connectivity and flexibility in entropic driven surface segregation of polymer-colloid mixtures.

Relative surface affinity between polymers and colloids is leveraged in many applications like filtration, adhesion, bio-sensing, etc. The surface affinity is governed by both enthalpic (relative interactions between the species and surface) and entropic (excluded volume) effects. Neglecting enthalpic effects, i.e. for purely athermal systems, entropy is the only driving force that controls the surface affinity of the species in a binary or multi-component mixture. Many intensive (relative size of colloids, chain length, equilibrium bond angle, chain flexibility) and extensive (confinement, temperature) factors can dramatically change the entropy of the system and thus enhance or decrease the surface affinities of the constituent species. In this article, we use coarse grained metropolis Monte Carlo simulations to delineate the role of these factors in entropic surface segregation in a binary mixture of polymers and colloids. At low number densities, excluded volume effects are negligible and we do not observe any entropic driven surface segregation. Therefore our system of interest is binary polymer-colloid mixtures at moderate to high number densities where excluded volume effects are predominant. Our results indicate that for flexible polymer chains, the surface is always enriched with colloids compared to polymers and this effect is enhanced for longer polymer chains. The configurational entropy of the flexible polymers is significantly reduced near the surface and therefore they prefer to stay in the bulk (away from the surface). However this behavior can be completely reversed by introducing a large degree of confinement and making the chains relatively rigid (less flexible). Our results show that polymer segregation of long stiff chains in slit pore geometry is driven by nematization near the surface while looping of polymers is observed under a large degree of confinement. We observe that for longer polymer chains with an equilibrium bond angle (θ = π), both confinement and chain stiffness enhance the surface segregation of polymers relative to colloids. However, the segregation behavior within a confinement is dependent on the polymer chain length. The surface segregation of polymers is dramatically decreased for chains with equilibrium bond angles independent of chain length and flexibility due to excluded volume effects and inefficient packing near the surface.

Hydrazide-Hydrazone Small Molecules as AIEgens: Illuminating Mitochondria in Cancer Cells.

Aggregation-induced-emission luminogens (AIEgens) have gained considerable attention as interesting tools for several biomedical applications, especially for bioimaging due to their brightness and photostability. Numerous AIEgens have been developed for lighting up the subcellular organelles to understand their forms and functions not only healthy but also unhealthy states, such as in cancer cells. However, there is lack of easily synthesizable, biocompatible small molecules for illuminating mitochondria (powerhouses) inside cells. To address this issue, an easy and short synthesis of new biocompatible hydrazide-hydrazone-based small molecules with remarkable aggregation-induced emission (AIE) properties is described. These small-molecule AIEgens showed hitherto unobserved AIE properties due to dual intramolecular H-bonding confirmed by theoretical calculation, pH- and temperature-dependent fluorescence and X-ray crystallographic studies. Confocal microscopy showed that these AIEgens were internalized into the HeLa cervical cancer cells without showing any cytotoxicity. One of the AIEgens was tagged with a triphenylphosphine (TPP) moiety, which successfully localized in the mitochondria of HeLa cells in a selective way compared to L929 noncancerous fibroblast cells. These unique hydrazide-hydrazone-based biocompatible AIEgens can serve as powerful tools to illuminate multiple subcellular organelles to elucidate their forms and functions in cancer cells for next-generation biomedical applications.

A "turn-off" red-emitting fluorophore for nanomolar detection of heparin.

A simple fluorophore bearing a diethylaminocoumarin donor and a pyridinium acceptor was synthesized and utilized for the ultra-sensitive detection of heparin. The synthesized dicationic push-pull coumarin derivative emits strongly in the red-region (665 nm) and detects nanomolar concentrations (14.8 nM to 148 nM) of heparin in HEPES buffer and FBS serum solutions. The dication exhibits excellent fluorescence selectivity and sensitivity towards heparin over its analogues such as chondroitin 4-sulfate (CS), hyaluronic acid (HA) and dextran. This fluorescence assay is a convenient, sensitive method for monitoring heparin levels in biological samples. These findings were confirmed using coarse-grained Monte Carlo simulations, which provide us with a rationale for the selective binding of heparin.

Sequence and entropy-based control of complex coacervates.

Biomacromolecules rely on the precise placement of monomers to encode information for structure, function, and physiology. Efforts to emulate this complexity via the synthetic control of chemical sequence in polymers are finding success; however, there is little understanding of how to translate monomer sequence to physical material properties. Here we establish design rules for implementing this sequence-control in materials known as complex coacervates. These materials are formed by the associative phase separation of oppositely charged polyelectrolytes into polyelectrolyte dense (coacervate) and polyelectrolyte dilute (supernatant) phases. We demonstrate that patterns of charges can profoundly affect the charge-charge associations that drive this process. Furthermore, we establish the physical origin of this pattern-dependent interaction: there is a nuanced combination of structural changes in the dense coacervate phase and a 1D confinement of counterions due to patterns along polymers in the supernatant phase.

Stability of proteins on hydrophilic surfaces.

The physical and chemical properties of solid substrates or surfaces critically influence the stability and activity of immobilized proteins such as enzymes. Reports of increased stability and activity of enzymes near/on surfaces as compared with those in solution abound; however, a mechanistic understanding is wanting. Simulations and experiments are used here to provide details toward such a mechanistic understanding. Experiments demonstrate increased activity of alcohol dehydrogenase (ADH) inside moderate hydrophilic mesopourous silica (SBA-15) pores but drastically decreased activity inside very hydrophilic NH2-SBA-15 surfaces as compared with that in solution. Also, the temperature stability of ADH was increased over that in solution when immobilized in a cavity with a mildly hydrophilic surface. Simulations confirm these experimental findings. Simulations calculated in the framework of a hydrophobic-polar (H-P) lattice model show increased thermal stability of a model 64-mer peptide on positive and zero curvature surfaces over that in solution. Peptides immobilized inside negative curvature cavities (concave) with hydrophilic surfaces exhibit increased stability only inside pores that are only 3-4 nm larger than the hydrodynamic radius of the peptide. Peptides are destabilized, however, when the surface hydrophilic character inside very small cavities/pores becomes large.

Surface-mediated protein disaggregation.

Preventing protein aggregation is of both biological and industrial importance. Interprotein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In this article, we show how surface chemistry and curvature can be tuned to mitigate these interprotein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that interprotein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose interprotein contacts to gain surface contacts, and thus the surface helps to reduce the interprotein interactions. Furthermore, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient at both reducing interprotein contacts and simultaneously retaining most of the native contacts probably as a result of confinement-induced stabilization.

Stability of proteins inside a hydrophobic cavity.

We study the effects of confinement and hydrophobicity of a spherical cavity on the structural and thermal stability of proteins in the framework of a hydrophobic-polar (HP) lattice model. We observe that a neutral confinement stabilizes the folded state of the protein by eliminating many of the open-chain conformations of the unfolded state. Hydrophobic confinement always destabilizes the protein because of protein-surface interactions. However, for moderate surface hydrophobicities, the protein remains stabilized relative to its state in free solution because of the dominance of entropic effects. These results are consistent with our experimental findings of (a) enhanced activity of alcohol dehydrogenase (ADH) when immobilized inside the essentially cylindrical pores of hydrophilic mesoporous silica (SBA-15) and (b) unaffected activity when immobilized inside weakly hydrophobic pores of methacrylate resin compared to its activity in free solution. In the same vein, our predictions are also consistent with the behavior of lysozyme and myoglobin in hydrophilic and hydrophobic SBA-15, which show qualitatively the same trends. Apparently, our results have validity across these very different enzymes, and we therefore suggest that confinement can be used to selectively improve enzyme performance.