Response of Foldable Protein Conformations to Non-Physiological Perturbations: Interplay of Thermal Factors and Confinement.

Technological advances frequently interface biomolecules with nanomaterials at non-physiological conditions, necessitating response characterization of key processes. Similar encounters are expected in cellular contexts. We report in silico investigations of the response of diverse protein conformational states to lowering of temperature and imposition of spatial constraints. Conformational states are represented by folded form of the Albumin binding domain (ABD) protein, its compact denatured form, and structurally disordered nascent folding elements. Data from extensive simulations are evaluated to elicit structural, thermodynamic and dynamic responses of the states and their associated environment. Analyses reveal alterations to folding propensity with reduced thermal energy and confinement, with signatures of trend reversal in highly disordered states. Across temperatures, confinement has restrictive effects on volume and energetic fluctuations, leading to narrowing of differences in isothermal compressibility (κ) and heat capacities (Cp). While excess (over ideal gas) entropy of the hydration layer marks dependence on the conformational state at bulk, confinement triggers erasure of differences. These observations are largely consistent with timescales of protein-water hydrogen bonding dynamics. The results implicate multi-factorial associations within a simple bio-nano complex. We expect the current study to motivate investigations of more biologically relevant interfaces towards mechanistic understanding and potential applications.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Conformational features of the Aß42 peptide monomer and its interaction with the surrounding solvent.

Accumulation of the amyloid beta (Aß) peptide in the brain is responsible for debilitating neurodegenerative diseases, such as Alzheimer's disease (AD). We have carried out atomistic molecular dynamics simulations of the full-length Aß42 peptide monomer with a wide range of conformations at room temperature. Efforts have been made to probe the conformational features of different segments of the peptide, namely the two terminal segments (N-term and C-term), the central hydrophobic regions (hp1 and hp2) and the central turn region joining hp1 and hp2, and their nonuniform influence on the spatial arrangements and binding energies of the surrounding water molecules. Our calculations reveal fluctuating conformations of the monomers with the formation and breaking of different secondary structural elements. In particular, it is noticed that the Aß monomers exhibit a propensity to either retain or transform into a helical form toward the N-term region and a ß-strand-like form near the C-term segment. Besides, heterogeneous conformational flexibility of the Aß monomers has been found to be correlated with the corresponding nonuniform entropy gains. Additionally, our calculation further reveals a heterogeneous hydration environment around the peptide. It is found that irrespective of the Aß peptide conformations and their nonuniform fluctuations, water molecules around the hydrophobic hp1 and hp2 segments are relatively weakly bound. This is an important observation, as in the presence of other monomers such weakly bound water molecules around hp1 and hp2 are expected to be easily displaced during the hydrophobic collapse that leads to Aß aggregation.

Unraveling origins of the heterogeneous curvature dependence of polypeptide interactions with carbon nanostructures.

Emerging nanotechnology has rapidly broadened interfacial prospects of biological molecules with carbon nanomaterials (CNs). A prerequisite for effectively harnessing such hybrid materials is a multi-faceted understanding of their complex interfacial interactions as functions of the physico-chemical characteristics and the surface topography of the individual components. In this article, we address the origins of the curvature dependence of polypeptide adsorption on CN surfaces (CNSs), a phenomenon bearing an acute influence upon the behavior and activity of CN-protein conjugates. Our benchmark molecular dynamics (MD) simulations with the amphiphilic full-length amyloid beta (Aß) peptide demonstrate that protein adsorption is strongest on the concave (inner) CN surface, weakest on the convex (outer) surface, and intermediary on the planar surface, in agreement with recent experimental reports. The curvature effects, however, are found to manifest non-uniformly between the amino acid subtypes. To understand the underlying interplay of the chemical nature of the amino acids and surface topography of the CNs, we performed high-level quantum chemical (QM) calculations with amino acid analogs (AAA) representing their five prominent classes, and convex, concave and planar CN fragments. Molecular electrostatic potential maps reveal pronounced curvature dependence in the mixing of electron densities, and a resulting variance in the stabilization of the non-covalently bound molecular complexes. Interestingly, our study revealed that the interaction trends of the high-level QM calculations were captured well by the empirical force field. The findings in this study have important bearing upon the design of carbon based bio-nanomaterials, and additionally, provide valuable insights into the accuracy of various computational techniques for probing non-bonded interfacial interactions.

Glycation induces conformational changes in the amyloid-ß peptide and enhances its aggregation propensity: molecular insights.

The cytotoxicity of the amyloid beta (Aß) peptide, implicated in the pathogenesis of Alzheimer's disease (AD), can be enhanced by its post-translational glycation, a series of non-enzymatic reactions with reducing sugars and reactive dicarbonyls. However, little is known about the underlying mechanisms that potentially enhance the cytotoxicity of the advanced glycation modified Aß. In this work, fully atomistic molecular dynamics (MD) simulations are exploited to obtain direct molecular insights into the process of early Aß self-assembly in the presence and absence of glycated lysine residues. Analyses of data exceeding cumulative timescales of 1 microsecond for each system reveal that glycation results in a stronger enthalpy of association between Aß monomers and lower conformational entropy, in addition to a sharp overall increase in the beta-sheet content. Further analyses reveal that the enhanced interactions originate, in large part, due to markedly stronger, as well as new, inter-monomer salt bridging propensities in the glycated variety. Interestingly, these conformational and energetic effects are broadly reflected in preformed protofibrillar forms of Aß small oligomers modified with glycation. Our combined results imply that glycation consolidates Aß self-assembly regardless of its point of occurrence in the pathway. They provide a basis for further mechanistic studies and therapeutic endeavors that could potentially result in novel ways of combating AGE related AD progression.

Aß self-association and adsorption on a hydrophobic nanosurface: competitive effects and the detection of small oligomers via electrical response.

Treatment of Alzheimer's disease (AD) is impeded by the lack of effective early diagnostic methods. Small, soluble Aß globulomers play a major role in AD neurotoxicity, and detecting their presence in aqueous fluids could lead to suitable sensors. We evaluate the adsorption behavior of small Aß oligomers on the surface of a single walled carbon nanotube of high curvature. While the intrinsic self-assembly propensity of Aß is markedly hindered by adsorption, the oligomeric units show high degrees of surface immobilization. Immobilized complexes are capable of oligomeric growth, but with a shifted monomer-oligomer equilibrium compared to the free states. In the presence of an ionic solution and suitable external electric fields, magnitudes of the current blockades are found to be sensitive to the oligomeric number of the adsorbed complex. However, this sensitivity gradually diminishes with increasing oligomeric size. The results provide a proof-of-concept basis for further investigations in the design of sensors for detecting the toxic small oligomers of Aß.

The non-uniform early structural response of globular proteins to cold denaturing conditions: a case study with Yfh1.

The mechanism of cold denaturation in proteins is often incompletely understood due to limitations in accessing the denatured states at extremely low temperatures. Using atomistic molecular dynamics simulations, we have compared early (nanosecond timescale) structural and solvation properties of yeast frataxin (Yfh1) at its temperature of maximum stability, 292 K (Ts), and the experimentally observed temperature of complete unfolding, 268 K (Tc). Within the simulated timescales, discernible "global" level structural loss at Tc is correlated with a distinct increase in surface hydration. However, the hydration and the unfolding events do not occur uniformly over the entire protein surface, but are sensitive to local structural propensity and hydrophobicity. Calculated infrared absorption spectra in the amide-I region of the whole protein show a distinct red shift at Tc in comparison to Ts. Domain specific calculations of IR spectra indicate that the red shift primarily arises from the beta strands. This is commensurate with a marked increase in solvent accessible surface area per residue for the beta-sheets at Tc. Detailed analyses of structure and dynamics of hydration water around the hydrophobic residues of the beta-sheets show a more bulk water like behavior at Tc due to preferential disruption of the hydrophobic effects around these domains. Our results indicate that in this protein, the surface exposed beta-sheet domains are more susceptible to cold denaturing conditions, in qualitative agreement with solution NMR experimental results.

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.

Molecular dynamics simulation studies of the structural response of an isolated Aß1-42 monomer localized in the vicinity of the hydrophilic TiO 2 surface.

We have probed the effect of a model hydrophilic surface, rutile TiO(2), on the full-length amyloid beta (Aß(1-42)) monomer using molecular dynamics simulations. The rutile surface brings about sharp changes in the peptide's intrinsic behavior in a distance-dependent manner. The intrinsic collapse of the peptide is disrupted, while the ß-sheet propensity is sharply enhanced with increased proximity to the surface. The results may have implications for Aß self-assembly and fibrillogenesis on hydrophilic surfaces and should be taken into consideration in the design of novel nanomaterials for perturbing amyloidogenic behavior.