Unearthing the inhibitory potential of phytochemicals from Lawsonia inermis L. and some drugs against dengue virus protein NS1: an in silico approach.

Dengue has received the status of an epidemic and endemic disease, with countless number of infections every year. Due to the unreliability of vaccines and non-specificity of drugs, it becomes necessary to find plant-based alternatives, with less harmful side effects. Lawsonia inermis L., is the sole source of dye, Mehendi. The rich repertoire of phytochemicals makes it useful, medicinally. The main objectives of the study are to explore the anti-dengue properties of the phytochemicals from Lawsonia inermis, and to shortlist potential candidates in curing the disease. Phytochemicals from the plant, and a set of drugs were screened and docked against NS1 protein, a less explored drug target, needed for maintenance of virus life cycle. Ligand screening and docking analysis concluded gallic acid, and chlorogenic acid to be good candidates, exhibiting high binding affinity and extensive interactions with the protein. From among the shortlisted drugs, only Vibegron showed effective binding affinity with NS1 protein with zero violations to the Lipinski's Rule of 5. Molecular dynamic simulations, executed for a time period of 100 nanoseconds, reveal the performance of a ligand within a solvated system. Chlorogenic and gallic acid, formed more stable and compact complexes with protein, with stable energy parameters and strong binding affinity. This was further validated with snapshots taken every 50 nanoseconds, showing no change in binding site between the ligand and protein, within the stipulated time frame. It was interesting to see that, a phenol (chlorogenic acid), served as a better drug candidate, against the NS1 protein.Communicated by Ramaswamy H. Sarma.

Topological Considerations in Biomolecular Condensation.

Biomolecular condensation and phase separation are increasingly understood to play crucial roles in cellular compartmentalization and spatiotemporal regulation of cell machinery implicated in function and pathology. A key aspect of current research is to gain insight into the underlying physical mechanisms of these processes. Accordingly, concepts of soft matter and polymer physics, the thermodynamics of mixing, and material science have been utilized for understanding condensation mechanisms of multivalent macromolecules resulting in viscoelastic mesoscopic supramolecular assemblies. Here, we focus on two topological concepts that have recently been providing key mechanistic understanding in the field. First, we will discuss how percolation provides a network-topology-related framework that offers an interesting paradigm to understand the complex networking of dense 'connected' condensate structures and, therefore, their phase behavior. Second, we will discuss the idea of entanglement as another topological concept that has deep roots in polymer physics and important implications for biomolecular condensates. We will first review some historical developments and fundamentals of these concepts, then we will discuss current advancements and recent examples. Our discussion ends with a few open questions and the challenges to address them, hinting at unveiling fresh possibilities for the modification of existing knowledge as well as the development of new concepts relevant to condensate science.

Molecular Origin of Internal Friction in Intrinsically Disordered Proteins.

Protein folding and dynamics are controlled by an interplay of thermal and viscosity effects. The effect of viscous drag through the solvent molecules is described by the classic Kramers theory in the high friction limit, which considers the dampening of the reactant molecules in the solution and quantifies the dependence of the reaction rate on the frictional drag. In addition to the external energy dissipation originating from the surrounding solvent molecules, there is an additional mode of internal energy dissipative force operative within the polypeptide chain reflecting the internal resistance of the chain to its conformational alterations. This dry, solvent-independent intrinsic frictional drag is termed internal friction. In the case of natively folded proteins, the physical origin of internal friction is primarily attributed to the intrachain interactions or other nonnative interactions in their compact states. However, the molecular origin of internal friction in intrinsically disordered proteins (IDPs) remains elusive.In this Account, we address this fundamental issue: what are the principal drivers of viscosity-independent (dry) friction in highly solvated, expanded, conformationally flexible, rapidly fluctuating IDPs that do not possess persistent intrachain interactions? IDPs exhibit diffusive conformational dynamics that is predominantly dominated by the segmental motion of the backbone arising due to the dihedral rotations in the Ramachandran Φ-Ψ space. The physical origin of friction in a complex biopolymeric system such as IDPs can be described by classic polymer models, namely, Rouse/Zimm models with internal friction. These one-dimensional models do not invoke torsional fluctuation components. Kuhn's classic description includes the connection between internal friction and microscopic dihedral hopping. Based on our time-resolved fluorescence anisotropy results, we describe that the sequence-dependent, collective, short-range backbone dihedral rotations govern localized internal friction in an archetypal IDP, namely, α-synuclein. The highly sensitive, residue-specific fluorescence depolarization kinetics offers a potent methodology to characterize and quantify the directional decorrelation engendered due to the short-range dihedral relaxation of the polypeptide backbone in the dihedral space. We utilized this characteristic relaxation time scale as our dynamic readout to quantify the site-specific frictional component. Our linear viscosity-dependent model of torsional relaxation time scale furnished a finite nonzero time constant at the zero solvent viscosity representing the solvent-independent internal friction. These results unveil the effect of the degree of dihedral restraining parameter on the internal friction component by showing that a restrained proline residue imparts higher torsional stiffness in the chain segments and, therefore, exhibits higher internal friction. This Account sheds light on the molecular underpinning of the sequence-specific internal friction in IDPs and will be of interest to unmask the role of internal friction in a diverse range of biomolecular processes involving binding-induced folding, allosteric interaction, protein misfolding and aggregation, and biomolecular condensation via phase separation.

Short-Range Backbone Dihedral Rotations Modulate Internal Friction in Intrinsically Disordered Proteins.

Protein folding and dynamics are governed by an intricate interplay of thermal and viscosity-mediated effects. The solvent viscosity contributes to the frictional drag in protein dynamics. In addition to this viscosity-dependent effect, there is also an intriguing viscosity-independent component that represents the intrinsic resistance of the polypeptide chain to changing its conformation. This solvent-independent component is termed internal friction. A longstanding question is what is the fundamental molecular origin of internal friction in highly solvated and rapidly fluctuating intrinsically disordered proteins (IDPs) devoid of any persistent intrachain interactions? Here, we present a unique case to directly demonstrate that sequence-specific backbone dihedral barriers control local internal friction in an archetypal IDP, namely, α-synuclein. We performed site-directed fluorescence depolarization kinetics using picosecond time-resolved fluorescence anisotropy measurements to directly observe the directional decorrelation arising due to short-range backbone torsional fluctuations in the dihedral space. A linear viscosity-dependent model of the dihedral relaxation time yielded a finite zero-viscosity intercept that corresponds to internal friction. Our site-specific dynamic readouts were able to detect localized sequence-specific frictional components that are otherwise skewed in viscosity-dependent long-range chain fluctuations. Our results revealed the presence of low internal friction in nonproline sequence segments. In contrast, a proline introduces torsional stiffness in the segment exhibiting high internal friction that can be compensated by a conformationally flexible glycine. Such an intriguing interplay of local dihedral dynamics can modulate sequence-dependent internal friction in a wide range of IDPs involved in a myriad of important events including folding, binding, assembly, and phase transitions.

Fluorescence Depolarization Kinetics Captures Short-Range Backbone Dihedral Rotations and Long-Range Correlated Dynamics of an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) do not autonomously fold into well-defined three-dimensional structures and are best described as a heterogeneous ensemble of rapidly interconverting conformers. It is challenging to elucidate their complex dynamic signatures using a single technique. In this study, we employed sensitive fluorescence depolarization kinetics by following picosecond time-resolved fluorescence anisotropy decays to directly capture the essential dynamical features of intrinsically disordered α-synuclein (α-syn) site-specifically labeled with thiol-active fluorophores. By utilizing a long-lifetime (≥10 ns) anisotropic label, we were able to discern three distinct rotational components of α-syn. The subnanosecond component represents the local wobbling-in-cone motion of the fluorophore, whereas the slower (∼1.4 ns) component corresponds to the short-range backbone dynamics governed by collective torsional fluctuations in the Ramachandran Φ-Ψ dihedral space. This backbone dihedral rotational time scale is sensitive to the local chain stiffness and slows down in the presence of an adjacent proline residue. We also observed a small-amplitude (≤10%) slower rotational correlation time (6-10 ns) that represents the long-range correlated dynamics involving a much longer segment of the polypeptide chain. These intrinsic dynamic signatures of IDPs will provide critical mechanistic underpinnings in a mosaic of biophysical phenomena involving internal friction, allosteric interactions, and phase separation.

Conformation-specific perturbation of membrane dynamics by structurally distinct oligomers of Alzheimer's amyloid-ß peptide.

The accumulation of toxic soluble oligomers of the amyloid-ß peptide (Aß) is a key step in the pathogenesis of Alzheimer's disease. There are mainly two conformationally distinct oligomers, namely, prefibrillar and fibrillar oligomers, that are recognized by conformation-specific antibodies, anti-amyloid oligomer antibody (A11) and anti-amyloid fibrillar antibody (OC), respectively. Previous studies have shown that the interaction of Aß oligomers with the lipid membrane is one of the key mechanisms of toxicity produced by Aß oligomers. However, the mechanism by which structurally distinct Aß oligomers interact with the lipid membrane remains elusive. In this work, we dissect the molecular mechanism underlying the interaction of structurally distinct Aß42 oligomers with the lipid membrane derived from the brain total lipid extract. Using picosecond time-resolved fluorescence spectroscopy, we show that the A11-positive Aß42 oligomers undergo a membrane-induced conformational change that promotes the deeper immersion of these oligomers into the lipid hydrocarbon region and results in an increase in the membrane micro-viscosity. In sharp contrast, OC-positive Aß42 oligomers interact with the lipid membrane via electrostatic interactions between the negatively-charged lipid headgroup and positively-charged residues of Aß42 without perturbing the membrane dynamics. We show that the two structurally distinct Aß42 oligomers demonstrating different interaction mechanisms with the lipid membrane eventually lead to the formation of typical amyloid fibrils. Our findings provide the mechanistic underpinning of the perturbation of lipid membranes by two conformationally distinct Aß42 oligomers and can be of prime importance in designing anti-Alzheimer's therapeutic agents targeting Aß-membrane interactions.

Excitation Energy Migration Unveils Fuzzy Interfaces within the Amyloid Architecture.

Amyloid fibrils are highly ordered nanoscopic protein aggregates comprising a cross-ß amyloid core and are associated with deadly human diseases. Structural studies have revealed the supramolecular architecture of a variety of disease-associated amyloids. However, the critical role of transient intermolecular interactions between the disordered polypeptide segments of protofilaments in directing the supramolecular structure and nanoscale morphology remains elusive. Here, we present a unique case to demonstrate that interchain excitation energy migration via intermolecular homo-Förster resonance energy transfer can decipher the architecture of amyloid fibrils of human α-synuclein. Site-specific homo-Förster resonance energy transfer efficiencies measured by fluorescence depolarization allowed us to construct a two-dimensional proximity correlation map that defines the supramolecular packing of α-synuclein within the fibrils. These studies captured unique heteroterminal cross talks between the fuzzy interprotofilament interfaces of the parallel-in-register amyloid spines. Our results will find applications in discerning the broader role of protein disorder and fuzziness in steering the distinct polymorphic amyloids that exhibit strain-specific disease phenotypes.

Energy migration captures membrane-induced oligomerization of the prion protein.

Excitation energy migration via homo-Förster resonance energy transfer (homo-FRET) can serve as an intermolecular proximity ruler within complex biomolecular assemblies. Here we present a unique case to demonstrate that energy migration can be a novel and sensitive readout to capture the membrane-mediated misfolding and oligomerization of the human prion protein (PrP), which is known to undergo an aberrant conformational conversion from an α-helical form into a self-propagating aggregated ß-rich state causing deadly transmissible neurodegenerative diseases. Using site-specific energy migration studies by monitoring steady-state and time-resolved fluorescence anisotropy of fluorescently-tagged PrP, we elucidate the molecular details of lipid membrane-induced oligomers. We show that the intrinsically disordered N-terminal segment is critical for lipid-induced conformational sequestration of PrP into higher-order, ß-rich oligomeric species that exhibit membrane permeabilization. Our results revealed that the N-terminal regions constitute the central core of the oligomeric architecture, whereas the distal C-terminal ends participate in peripheral association with the lipid membrane. Our study will find applications in the sensitive detection and in the structural characterization of membrane-induced protein misfolding and aggregation in a variety of deadly amyloid diseases.

Functional conservation of CYCLOPS in crack entry legume Arachis hypogaea.

Root nodule symbiosis in legumes is established following interaction of compatible rhizobia that activates an array of genes, commonly known as symbiotic-pathway, resulting in nodule development. In model legumes, bacterial entry mainly occurs through infection thread involving the expression of transcription factor CYCLOPS/IPD3. Here we show the functional analysis of AhCYCLOPS in Arachis hypogaea where bacteria invade roots through epidermal cracks. Exploiting significant cross-species domain conservation, trans-complementation experiments involving ectopic expression of AhCYCLOPS in transgenic hairy-roots of Medicago truncatula ipd3 mutants resulted in functional complementation of Medicago nodules. Moreover, native promoter of AhCYCLOPS was sufficient for this cross-species complementation irrespective of the different modes of infection of roots by rhizobia and nodule ontology. To unravel the role of AhCYCLOPS during 'crack-entry' nodulation in A. hypogaea, RNAi of AhCYCLOPS was performed which resulted in delayed nodule inception followed by drastic reduction in nodule number on transgenic hairy-roots. The infection zone of a significant number of RNAi nodules showed presence of infected cells with enlarged nucleus and rod shaped undifferentiated bacteria. Expression analysis showed downregulation of several nodulation responsible effectors endorsing the compromised condition of RNAi roots. Together, the results indicated that AhCYCLOPS plays an important role in A. hypogaea nodule development.

Studying backbone torsional dynamics of intrinsically disordered proteins using fluorescence depolarization kinetics.

Intrinsically disordered proteins (IDPs) do not autonomously adopt a stable unique 3D structure and exist as an ensemble of rapidly interconverting structures. They are characterized by significant conformational plasticity and are associated with several biological functions and dysfunctions. The rapid conformational fluctuation is governed by the backbone segmental dynamics arising due to the dihedral angle fluctuation on the Ramachandran φ- ψ conformational space. We discovered that the intrinsic backbone torsional mobility can be monitored by a sensitive fluorescence readout, namely fluorescence depolarization kinetics, of tryptophan in an archetypal IDP such as α-synuclein. This methodology allows us to map the site-specific torsional mobility in the dihedral space within picosecond-nanosecond time range at a low protein concentration under the native condition. The characteristic timescale of ~1.4 ns, independent of residue position, represents collective torsional dynamics of dihedral angles (φ and ψ) of several residues from tryptophan and is independent of overall global tumbling of the protein. We believe that fluorescence depolarization kinetics methodology will find broad application to study both short-range and long-range correlated motions, internal friction, binding-induced folding, disorder-to-order transition, misfolding and aggregation of IDPs.