Helical reorganization in the context of membrane protein folding: Insights from simulations with bacteriorhodopsin (BR) fragments.

Membrane protein folding is distinct from folding of soluble proteins. Conformational acquisition in major membrane protein subclasses can be delineated into insertion and folding processes. An exception to the "two stage" folding, later developed to "three stage" folding, is observed within the last two helices in bacteriorhodopsin (BR), a system that serves as a model membrane protein. We employ a reductionist approach to understand interplay of molecular factors underlying the apparent defiance. Leveraging available solution NMR structures, we construct, sample in silico, and analyze partially (PIn) and fully inserted (FIn) BR membrane states. The membrane lateral C-terminal helix (CH) in PIn is markedly prone to transient structural distortions over microsecond timescales; a disorder prone region (DPR) is thereby identified. While clear transmembrane propensities are not acquired, the distortions induce alterations in local membrane curvature and area per lipid. Importantly, energetic decompositions reveal that overall, the N-terminal helix (NH) is thermodynamically more stable in the PIn. Higher overall stability of the FIn arises from favorable interactions between the NH and the CH. Our results establish lack of spontaneous transition of the PIn to the FIn, and attributes their partitioning to barriers that exceed those accessible with thermal fluctuations. This work paves the way for further detailed studies aimed at determining the thermo-kinetic roles of the initial five helices, or complementary external factors, in complete helical folding and insertion in BR. We comment that complementing such efforts with the growing field of machine learning assisted energy landscape searches may offer unprecedented insights.

Thermal Sensitivity of the Enzymatic Activity of ß-Glucosidase: Simulations Lend Mechanistic Insights into Experimental Observations.

A crucial prerequisite for industrial applications of enzymes is the maintenance of specific activity across wide thermal ranges. ß-Glucosidase (EC 3.2.1.21) is an essential enzyme for converting cellulose in biomass to glucose. While the reaction mechanisms of ß-glucosidases from various thermal ranges (hyperthermophilic, thermophilic, and mesophilic) are similar, the factors underlying their thermal sensitivity remain obscure. The work presented here aims to unravel the molecular mechanisms underlying the thermal sensitivity of the enzymatic activity of the ß-glucosidase BglB from the bacterium Paenibacillus polymyxa. Experiments reveal a maximum enzymatic activity at 315 K, with a marked decrease in the activity below and above this temperature. Employing in silico simulations, we identified the crucial role of the active site tunnel residues in the thermal sensitivity. Specific tunnel residues were identified via energetic decomposition and protein-substrate hydrogen bond analyses. The experimentally observed trends in specific activity with temperature coincide with variations in overall binding free energy changes, showcasing a predominantly electrostatic effect that is consistent with enhanced catalytic pocket-substrate hydrogen bonding (HB) at Topt. The entropic advantage owing to the HB substate reorganization was found to facilitate better substrate binding at 315 K. This study elicits molecular-level insights into the associative mechanisms between thermally enabled fluctuations and enzymatic activity. Crucial differences emerge between molecular mechanisms involving the actual substrate (cellobiose) and a commonly employed chemical analogue. We posit that leveraging the role of fluctuations may reveal unexpected insights into enzyme behavior and offer novel paradigms for enzyme engineering.

Base pair compositional variability influences DNA structural stability and tunes hydration thermodynamics and dynamics.

DNA deformability and differential hydration are crucial determinants of biological processes ranging from genetic material packaging to gene expression; their associative details, however, remain inadequately understood. Herein, we report investigations of the dynamic and thermodynamic responses of the local hydration of a variety of base pair sequences. Leveraging in silico sampling and our in-house analyses, we first report the local conformational propensity of sequences that are either predisposed toward the canonical A- or B-conformations or are restrained to potential transitory pathways. It is observed that the transition from the unrestrained A-form to the B-form leads to lengthwise structural deformation. The insertion of intermittent -(CG)- base pairs in otherwise homogeneous -(AT)- sequences bears dynamical consequences for the vicinal hydration layer. Calculation of the excess (pair) entropy suggests substantially higher values of hydration water surrounding A conformations over the B- conformations. Applying the Rosenfeld approximation, we project that the diffusivity of water molecules proximal to canonical B conformation is least for the minor groove of the canonical B-conformation. We determine that structure, composition, and conformation specific groove dimension together influence the local hydration characteristics and, therefore, are expected to be important determinants of biological processes.

Conformational ensemble of the NSP1 CTD in SARS-CoV-2: Perspectives from the free energy landscape.

The nonstructural protein-1 (NSP1) of the severe acute respiratory syndrome-associated coronavirus 2 plays a crucial role in the translational shutdown and immune evasion inside host cells. Despite its known intrinsic disorder, the C-terminal domain (CTD) of NSP1 has been reported to form a double α-helical structure and block the 40S-ribosomal channel for mRNA translation. Experimental studies indicate that NSP1 CTD functions independently from the globular N-terminal region separated with a long linker domain, underscoring the necessity of exploring the standalone conformational ensemble. In this contribution, we utilize exascale computing resources to yield unbiased molecular dynamics simulation of NSP1 CTD in all-atom resolution starting from multiple initial seed structures. A data-driven approach elicits collective variables (CVs) that are significantly superior to conventional descriptors in capturing the conformational heterogeneity. The free energy landscape as a function of the CV space is estimated using the modified expectation maximized molecular dynamics. Originally developed by us for small peptides, here, we establish the efficacy of expectation maximized molecular dynamics in conjunction with data-driven CV space for a more complex and relevant biomolecular system. The results reveal the existence of two disordered metastable populations in the free energy landscape that are separated from the conformation resembling ribosomal subunit bound state by high kinetic barriers. Chemical shift correlation and secondary structure analysis capture significant differences among key structures of the ensemble. Altogether, these insights can underpin drug development studies and mutational experiments that help induce population shifts to alter translational blocking and understand its molecular basis in further detail.

Theoretical Chemistry Symposium 2021.

The editorial paints a brief history of the conference - Theoretical Chemistry Symposium, organized by eminent theoretical and computational chemists from India.

Efficient Interrogation of the Kinetic Barriers Demarcating Catalytic States of a Tyrosine Kinase with Optimal Physical Descriptors and Mixture Models.

Computer simulations are increasingly used to access thermo-kinetic information underlying structural transformation of protein kinases. Such information are necessary to probe their roles in disease progression and interactions with drug targets. However, the investigations are frequently challenged by forbiddingly high computational expense, and by the lack of standard protocols for the design of low dimensional physical descriptors that encode system features important for transitions. Here, we consider the demarcating characteristics of the different states of Abelson tyrosine kinase associated with distinct catalytic activity to construct a set of physically meaningful, orthogonal collective variables that preserve the slow modes of the system. Independent sampling of each metastable state is followed by the estimation of global partition function along the appropriate physical descriptors using the modified Expectation Maximized Molecular Dynamics method. The resultant free energy barriers are in excellent agreement with experimentally known rate-limiting dynamics and activation energy computed with conventional enhanced sampling methods. We discuss possible directions for further development and applications.

Effects of External Perturbations on Protein Systems: A Microscopic View.

Protein folding can be viewed as the origami engineering of biology resulting from the long process of evolution. Even decades after its recognition, research efforts worldwide focus on demystifying molecular factors that underlie protein structure-function relationships; this is particularly relevant in the era of proteopathic disease. A complex co-occurrence of different physicochemical factors such as temperature, pressure, solvent, cosolvent, macromolecular crowding, confinement, and mutations that represent realistic biological environments are known to modulate the folding process and protein stability in unique ways. In the current review, we have contextually summarized the substantial efforts in unveiling individual effects of these perturbative factors, with major attention toward bottom-up approaches. Moreover, we briefly present some of the biotechnological applications of the insights derived from these studies over various applications including pharmaceuticals, biofuels, cryopreservation, and novel materials. Finally, we conclude by summarizing the challenges in studying the combined effects of multifactorial perturbations in protein folding and refer to complementary advances in experiment and computational techniques that lend insights to the emergent challenges.

Dynamical Manifestations of Supercooling in Amyloid Hydration.

The effect of extreme temperature on amyloidogenic species remains sparsely explored. In a recent study (J. Phys. Chem. Lett., 2019, 10, (10)), we employed exhaustive molecular dynamics simulations to explore the cold thermal response of a putative small amyloid oligomer and to elicit the role of solvent modulation. Herein, we investigate the dynamical response of the hydration waters of the oligomer within the supercooled states. Using NMR-based formalism, we delineate the entropic response in terms of the side-chain conformational entropy that corroborates the weakening of the hydrophobic core with lowering of temperature. The translational dynamics of the protein and hydration waters reveal the coupling of protein dynamical fluctuations with solvent dynamics under supercooled conditions. Probing the translational motion as a space-time correlation indicates glassy dynamics exhibited by hydration waters in the supercooled regime. Caging of the water molecules with lowering of temperature and the resultant hopping dynamics are reflected in the longer ß-relaxation timescales of translational motion. Furthermore, we utilized mode-coupling theory (MCT) and derived the ideal glass transition temperature from translational and rotational dynamics, around ∼196 and 209 K, respectively. Interestingly, rotational motion in the supercooled regime deviates from the MCT law, exhibits Arrhenius motion, and marks a fragile-to-strong crossover at 227 K. The low-frequency vibrational modes also coincide with the dynamical transition. This exposition lends dynamical insights into the hydration coupling of an amyloid aggregate under cryogenic conditions.

Hydroxy-Porphyrin as an Effective, Endogenous Molecular Clamp during Early Stages of Amyloid Fibrillization.

Amyloid fibril formation of proteins is of great concern in neurodegenerative disease and can be detrimental to the storage and stability of biologics. Recent evidence suggests that insulin fibril formation reduces the efficacy of type II diabetes management and may lead to several complications. To develop anti-amyloidogenic compounds of endogenous origin, we have utilized the hydrogen bond anchoring, π stacking ability of porphyrin, and investigated its role on the inhibition of insulin amyloid formation. We report that hydroxylation and metal removal from the heme moiety yields an excellent inhibitor of insulin fibril formation. Thioflavin T, tyrosine fluorescence, Circular Dichorism (CD) spectroscopy, Field emission scanning electron microscopy (FESEM) and molecular dynamics (MD) simulation studies suggest that hematoporphyrin (HP) having hydrogen bonding ability on both sides is a superior inhibitor compared to hemin and protoporphyrin (PP). Experiments with hen egg white lysozyme (HEWL) amyloid fibril formation also validated the efficacy of endogenous porphyrin based small molecules. Our results will help to decipher a general therapeutic strategy to counter amyloidogenesis.

The protein hydration layer in high glucose concentration: Dynamical responses in folded and intrinsically disordered dimeric states.

This exposition reveals the effect of glucose as a molecular crowder on the solvent environment in proximity of the protein surface in putative folded (Ubiquitin) and intrinsically disordered (dimeric Amyloid beta) states. Atomistic simulations reveal markedly higher structural perturbation in the disordered systems due to crowding effects, while the folded state retains overall structural fidelity. Key hydrophobic contacts in the disordered dimer are lost. However, glucose induced crowding results in elevated hydration on surfaces of both protein systems. Despite evident differences in their structural responses, the hydration layer of both the folded and disordered states display a distinct enhancement in lifetimes of mean residence and rotational relaxation under the hyperglycemic conditions. The results are crucial in the light of emergent co-solvent induced biological phenomena in crowded media.

Hydration of a small protein under carbon nanotube confinement: Adsorbed substates induce selective separation of the dynamical response.

The co-involvement of biological molecules and nanomaterials has increasingly come to the fore in modern-day applications. While the "bio-nano" (BN) interface presents physico-chemical characteristics that are manifestly different from those observed in isotropic bulk conditions, the underlying molecular reasons remain little understood; this is especially true of anomalies in interfacial hydration. In this paper, we leverage atomistic simulations to study differential adsorption characteristics of a small protein on the inner (concave) surface of a single-walled carbon nanotube whose diameter exceeds dimensions conducive to single-file water movement. Our findings indicate that the extent of adsorption is decided by the degree of foldedness of the protein conformational substate. Importantly, we find that partially folded substates, but not the natively folded one, induce reorganization of the protein hydration layer into an inner layer water closer to the nanotube axis and an outer layer water in the interstitial space near the nanotube walls. Further analyses reveal sharp dynamical differences between water molecules in the two layers as observed in the onset of increased heterogeneity in rotational relaxation and the enhanced deviation from Fickian behavior. The vibrational density of states reveals that the dynamical distinctions are correlated with differences in crucial bands in the power spectra. The current results set the stage for further systematic studies of various BN interfaces vis-à-vis control of hydration properties.

Expectation maximized molecular dynamics: Toward efficient learning of rarely sampled features in free energy surfaces from unbiased simulations.

Biophysical processes often encounter high energy transition states that lie in regions of the free energy landscape (FEL) inaccesible to conventional molecular dynamics simulations. Various enhanced sampling methods have been developed to handle the inherent quasi-nonergodicity, either by adding a biasing potential to the underlying Hamiltonian or by forcing the transitions with parallel tempering. However, when attempting to probe systems of increasing complexity with limited computational resources, there arises an imminent need for fast and efficient FEL exploration with sufficient accuracy. Herein, we present a computationally efficient algorithm based on statistical inference for fast estimation of key features in the two-dimensional FEL. Unlike conventional enhanced sampling methods, this newly developed method avoids direct sampling of high free energy states. Rather, the transition states connecting metastable regions of comparable free energies are estimated using Bayesian likelihood maximization. Furthermore, the method incorporates a tunable self-feedback mechanism with classical molecular dynamics for preventing unnecessary sampling that no more effectively contributes to the underlying distributions of metastable states. We have applied this novel protocol in three independent case studies and compared the results against a conventional method. We conclude with the scope of further developments for improved accuracy of the new method and its generalization toward estimation of features in more complex FELs.

Amyloid-ß regulates gap junction protein connexin 43 trafficking in cultured primary astrocytes.

Altered expression and function of astroglial gap junction protein connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). Although earlier studies have examined the effect of increased ß-amyloid (Aß) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aß modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aß can alter Cx43 gap junctions. We show that Aß25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aß25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulum-associated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aß25-35-exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aß-induced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aß25-35-exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aß25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aß can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aß modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.

Disulfide Reduction Allosterically Destabilizes the ß-Ladder Subdomain Assembly within the NS1 Dimer of ZIKV.

The Zika virus (ZIKV) was responsible for a recent debilitating epidemic that till date has no cure. A potential way to reduce ZIKV virulence is to limit the action of the nonstructural proteins involved in its viral replication. One such protein, NS1, encoded as a monomer by the viral genome, plays a major role via symmetric oligomerization. We examine the homodimeric structure of the dominant ß-ladder segment of NS1 with extensive all atom molecular dynamics. We find it stably bounded by two spatially separated interaction clusters (C1 and C2) with significant differences in the nature of their interactions. Four pairs of distal, intramonomeric disulfide bonds are found to be coupled to the stability, local structure, and wettability of the interfacial region. Symmetric reduction of the intramonomeric disulfides triggers marked dynamical heterogeneity, interfacial wettability, and asymmetric salt-bridging propensity. Harnessing the model-free Lipari-Szabo based formalism for estimation of conformational entropy (Sconf), we find clear signatures of heterogeneity in the monomeric conformational entropies. The observed asymmetry, very small in the unperturbed state, expands significantly in the reduced states. This allosteric effect is most noticeable in the electrostatically bound C2 cluster that underlies the greatest stability in the unperturbed state. Allosteric induction of conformational and thermodynamic asymmetry is expected to affect the pathways leading to symmetric higher-ordered oligomerization, and thereby affect crucial replication pathways.

Nanoscale Interplay of Membrane Composition and Amyloid Self-Assembly.

Cell membranes are complex assemblies of lipids and proteins exhibiting lipid compositional heterogeneity between the inner and outer leaflets of the bilayer. Aberrant protein aggregation, implicated in a number of neurodegenerative diseases including Alzheimer's, is known to result in both extracellular and intracellular deposits with divergent pathophysiological effects. Mounting evidence substantiates membrane-mediated amyloid effects and indicates membrane composition, particularly gangliosides, as a plausible factor influencing the fibrillation process. By employing exhaustive molecular dynamics simulations using a coarse-grained model, we probed the assembly behavior of amyloidogenic Aß(12-28) peptides on the chemically heterogeneous extracellular (outer) and cytosolic (inner) leaflets of a mammalian plasma membrane. Our results indicate that the compositional nature of the membrane has a crucial impact on the peptide self-assembly. Peptide oligomerization is hindered on the outer leaflet relative to the inner leaflet due to a competition between interpeptide and peptide-membrane interactions, resulting in higher population of smaller oligomers. The weaker associations among peptides on the outer membrane can be attributed to the favorable interactions of the peptides with gangliosides (GM) that characterize the extracellular membrane. At a higher peptide:GM ratio, we observe enhanced nanoclustering of GM lipids mediated by preferential GM-Aß binding. Interaction between peptide and GM further impacts local membrane curvature; there is a concomitant loss in membrane concavity due to looser GM packing. Our simulations provide molecular insights into the role of membrane composition on Aß aggregation and lend credence to earlier reports of ganglioside-mediated Aß aggregation in the outer membrane. We also demonstrate the effects of local peptide assemblies on the membrane structure and dynamics.

Influence of crowding and surfaces on protein amyloidogenesis: A thermo-kinetic perspective.

The last few decades have irreversibly implicated protein self-assembly and aggregation leading to amyloid fibril formation in proteopathies that include several neurodegenerative diseases. Emerging studies recognize the importance of eliciting the pathways leading to protein aggregation in the context of the crowded intracellular environment rather than in conventional in vitro conditions. It is found that crowded environments can have acceleratory as well as inhibitory effects on protein aggregation, depending on the interplay of underlying factors on the crucial rate limiting steps. The aggregation mechanism and transient species formed along the pathway are further altered when they interface with natural and artificial surfaces in the cellular milieu. An increasing number of studies probe the autocatalytic nature of amyloid surfaces as well as membrane bilayer effects on amyloidogenesis. Moreover, exposure to modern nanosurfaces via nanomedicines and other sources potentially invokes beneficial or deleterious biological response that needs rigorous investigation. Mounting evidences indicate that nanoparticles can either promote or impede amyloid aggregation, spurring efforts to tune their interactions for developing effective anti-amyloid strategies. Mechanistic insights into nanoparticle mediated aggregation pathways are therefore crucial for engineering anti-amyloid nanoparticle strategies that are biocompatible and sustainable. This review is a compilation of studies that contribute to the current understanding of the altering effects of molecular crowding as well as natural and artificial surfaces on protein amyloidogenesis.

The Cold Thermal Response of an Amyloid Oligomer Differs from Typical Globular Protein Cold Denaturation.

In contrast with the general behavior of folded proteins, the cold thermal response of amyloid assemblies is difficult to elicit with simple models. We exploit exhaustive simulations to evaluate the thermal response of a barrel-shaped model amyloid oligomer, with a distinct hydrophobic core akin to that of folded proteins. Cumulative thermal data over the range of 210-483 K indicate a sharp inflection and rise in structural stability as the temperature is decreased below the melting temperature of the water model. This is not commensurate with the equilibrium free energy profile obtained with core packing as the order parameter. However, energetic analyses and the size of their fluctuations indicate the crucial role of hydration in mediating structural transitions, beyond the expected temperature-dependent hydrophobic effect. Structural ordering of the hydration layer over bulk water is maximized at the transition and vanishes at high temperatures. This is a first direct demonstration of the microscopic influence of hydration water on the low-temperature response of an amyloid assembly close to the cryo-regime.

Cosolvent Impurities in SWCNT Nanochannel Confinement: Length Dependence of Water Dynamics Investigated with Atomistic Simulations.

The advent of nanotechnology has seen a growing interest in the nature of fluid flow and transport under nanoconfinement. The present study leverages fully atomistic molecular dynamics (MD) simulations to study the effect of nanochannel length and intrusion of molecules of the organic solvent, hexafluoro-2-propanol (HFIP), on the dynamical characteristics of water within it. Favorable interactions of HFIP with the nanochannels comprised of single-walled carbon nanotubes traps them over time scales greater than 100 ns, and confinement confers small but distinguishable spatial redistribution between neighboring HFIP pairs. Water molecules within the nanochannels show clear signatures of dynamical slowdown relative to bulk water even for pure systems. The presence of HFIP causes further rotational and translational slowdown in waters when the nanochannel dimension falls below a critical length of 30 Å. The enhanced slowdown in the presence of HFIP is quantified from characteristic relaxation parameters and diffusion coefficients in the absence and presence of HFIP. It is finally seen that the net flow of water between the ends of the nanochannel shows a decreasing dependence with nanochannel length only when the number of HFIP molecules is small. These results lend insights into devising ways of modulating solvent properties within nanochannels with cosolvent impurities.

Structural and dynamics studies of the TetR family protein, CprB from Streptomyces coelicolor in complex with its biological operator sequence.

In Streptomycetes, tetracycline repressor family of transcription regulators (TetR-FTRs) controls various biological processes including antibiotic biosynthesis, cellular morphology and innate resistance. Here, we focus on understanding the structural basis of transcription regulation by CprB, a member of TetR-FTRs from S. coelicolor. CprB is implicated as a receptor of γ-butyrolactones, a class of quorum sensing molecules, responsible for initiating secondary metabolic pathways. In order to understand the molecular mechanism of DNA recognition, the X-ray structure of CprB in complex with its biological relevant operator sequence was solved to a resolution of 3.95Å. Furthermore, to refine and compliment the results, atomistic molecular dynamics simulations were carried out using the X-ray structure as the template. The studies reveal that CprB binds to DNA as dimer of dimers with this mode of interaction results in minimal distortion in the DNA, enabling these proteins to recognize multiple sequences with varying affinity. Another crucial finding from our simulation results was that the positively charged N-terminal arm of CprB brings extra stability to the protein-DNA complex by interacting with the minor-groove of the DNA and anchoring itself to the phosphate backbone. Corroborating electrophoretic mobility shift assay and fluorescence anisotropy experiments showed that the mutant ΔN6-CprB exhibited about 7-8 fold reduced DNA binding. Comparison with other TetR-FTRs reveals that this strategy is also employed by over 25% of TetR-FTRs, where N-terminal anchoring mechanism is used to enhance selectivity for a particular DNA sequence.

Influence of Hyperglycemic Conditions on Self-Association of the Alzheimer's Amyloid ß (Aß1-42) Peptide.

Clinical studies have identified a correlation between type-2 diabetes mellitus and cognitive decrements en route to the onset of Alzheimer's disease (AD). Recent studies have established that post-translational modifications of the amyloid ß (Aß) peptide occur under hyperglycemic conditions; particularly, the process of glycation exacerbates its neurotoxicity and accelerates AD progression. In view of the assertion that macromolecular crowding has an altering effect on protein self-assembly, it is crucial to characterize the effects of hyperglycemic conditions via crowding on Aß self-assembly. Toward this purpose, fully atomistic molecular dynamics simulations were performed to study the effects of glucose crowding on Aß dimerization, which is the smallest known neurotoxic species. The dimers formed in the glucose-crowded environment were found to have weaker associations as compared to that of those formed in water. Binding free energy calculations show that the reduced binding strength of the dimers can be mainly attributed to the overall weakening of the dispersion interactions correlated with substantial loss of interpeptide contacts in the hydrophobic patches of the Aß units. Analysis to discern the differential solvation pattern in the glucose-crowded and pure water systems revealed that glucose molecules cluster around the protein, at a distance of 5-7 Å, which traps the water molecules in close association with the protein surface. This preferential exclusion of glucose molecules and resulting hydration of the Aß peptides has a screening effect on the hydrophobic interactions, which in turn diminishes the binding strength of the resulting dimers. Our results imply that physical effects attributed to crowded hyperglycemic environments are incapable of solely promoting Aß self-assembly, indicating that further mechanistic studies are required to provide insights into the self-assembly of post-translationally modified Aß peptides, known to possess aggravated toxicity, under these conditions.