Molecular Insights into the Conformational and Binding Behaviors of Human Serum Albumin Induced by Surface-Active Ionic Liquids.

Extensive research has been carried out to investigate the stability and function of human serum albumin (HSA) when exposed to surface-active ionic liquids (SAILs) with different head groups (imidazolium, morpholinium, and pyridinium) and alkyl chain lengths (ranging from decyl to tetradecyl). Analysis of the protein fluorescence spectra indicates noticeable changes in the secondary structure of HSA with varying concentrations of all SAILs tested. Helicity calculations based on the Fourier transform infrared (FTIR) data show that HSA becomes more organized at the micellar concentration of SAILs, leading to an increased protein activity at this level. Small-angle neutron scattering (SANS) data confirm the formation of a bead-necklace structure between the SAILs and HSA. Atomistic molecular dynamics (MD) simulation results identify several hotspots on the protein surface for interaction with SAIL, which results in the modulation of protein conformational fluctuation and stability. Furthermore, fluorescence resonance energy transfer (FRET) experiments with the intramolecular charge transfer (ICT) probe trans-ethyl p-(dimethylamino) cinnamate (EDAC) demonstrate that higher alkyl chain lengths and SAIL concentrations result in a significantly increased energy transfer efficiency. The findings of this study provide a detailed molecular-level understanding of how the protein structure and function are affected by the presence of SAILs, with potential implications for a wide range of applications involving protein-SAIL composite systems.

Leveraging Bidirectional Nature of Allostery To Inhibit Protein-Protein Interactions (PPIs): A Case Study of PCSK9-LDLR Interaction.

The protein PCSK9 (proprotein convertase subtilisin/Kexin type 9) negatively regulates the recycling of LDLR (low-density lipoprotein receptor), leading to an elevated plasma level of LDL. Inhibition of PCSK9-LDLR interaction has emerged as a promising therapeutic strategy to manage hypercholesterolemia. However, the large interaction surface area between PCSK9 and LDLR makes it challenging to identify a small molecule competitive inhibitor. An alternative strategy would be to identify distal cryptic sites as targets for allosteric inhibitors that can remotely modulate PCSK9-LDLR interaction. Using several microseconds long molecular dynamics (MD) simulations, we demonstrate that on binding with LDLR, there is a significant conformational change (population shift) in a distal loop (residues 211-222) region of PCSK9. Consistent with the bidirectional nature of allostery, we establish a clear correlation between the loop conformation and the binding affinity with LDLR. Using a thermodynamic argument, we establish that the loop conformations predominantly present in the apo state of PCSK9 would have lower LDLR binding affinity, and they would be potential targets for designing allosteric inhibitors. We elucidate the molecular origin of the allosteric coupling between this loop and the LDLR binding interface in terms of the population shift in a set of salt bridges and hydrogen bonds. Overall, our work provides a general strategy toward identifying allosteric hotspots: compare the conformational ensemble of the receptor between the apo and bound states of the protein and identify distal conformational changes, if any. The inhibitors should be designed to bind and stabilize the apo-specific conformations.

Conformational Effects on the Absorption Spectra of Phytochromes.

Bacteriophytochrome is a photoreceptor protein that contains the biliverdin (BV) chromophore as its active component. The spectra of BV upon mutation remain remarkably unchanged, as far as spectral positions are concerned. This points toward the minimal effect of electrostatic effects on the electronic structure of the chromophore. However, the relative intensities of the Q and Soret bands of the chromophore change dramatically upon mutation. In this work, we delve into the molecular origin of this unusual intensity modulation. Using extensive classical MD and QM/MM calculations, we show that due to mutation, the conformational population of the chromophore changes significantly. The noncovalent interactions, especially the stacking interactions, lead to extra stabilization of the cyclic form in the D207H mutated species as opposed to the open form in the wild-type BV. Thus, unlike the commonly observed direct electrostatic effect on the spectral shift, in the case of BV the difference observed is in varying intensities, and this in turn is driven by a conformational shift due to enhanced stacking interaction.

Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI.

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

Enabling Broader Adoption of Biocatalysis in Organic Chemistry.

Biocatalysis is becoming an increasingly impactful method in contemporary synthetic chemistry for target molecule synthesis. The selectivity imparted by enzymes has been leveraged to complete previously intractable chemical transformations and improve synthetic routes toward complex molecules. However, the implementation of biocatalysis in mainstream organic chemistry has been gradual to this point. This is partly due to a set of historical and technological barriers that have prevented chemists from using biocatalysis as a synthetic tool with utility that parallels alternative modes of catalysis. In this Perspective, we discuss these barriers and how they have hindered the adoption of enzyme catalysts into synthetic strategies. We also summarize tools and resources that already enable organic chemists to use biocatalysts. Furthermore, we discuss ways to further lower the barriers for the adoption of biocatalysis by the broader synthetic organic chemistry community through the dissemination of resources, demystifying biocatalytic reactions, and increasing collaboration across the field.

Efficient light harvesting in self-assembled organic luminescent nanotubes.

Luminescent organic nanotubes derived from the co-assembly of cyanostilbene (CS) based cationic supramolecular polymers and bio-polyanion heparin, a known anticoagulant, have been utilized as highly efficient FRET (fluorescence resonance energy transfer) donors in aqueous media resulting in amplified acceptor emission in the orange-red and near-infrared (NIR). Energy transfer efficiencies higher than 80% and an ultra-high antenna effect of 150 were achieved even at high donor/acceptor ratios (500 : 1-100 : 1) translating to emission quenching of several hundred donors by one acceptor. Utilizing the temperature responsiveness of the FRET process, these systems were employed as ratiometric emission thermometers in the temperature range 20-90 °C. Moreover, the energy transfer was very effective in solid and polymer films. This allowed us to generate multi-color emissions ranging from blue to red including white light in solution as well as in solid and polymer films.

Molecular Thermodynamic Origin of Substrate Promiscuity in the Enzyme Laccase: Toward a Broad-Spectrum Degrader of Dye Effluents.

Industrial dye effluents have emerged as significant health hazard. Laccases found in white rot fungi can degrade an assortment of dyes. Here, we explore the molecular thermodynamic origin of the substrate promiscuity in laccases using a combination of steady-state UV-visible absorption spectroscopy, molecular docking, and molecular dynamics (MD) simulation studies on the interaction of laccase with five dye molecules with varying charge, size, and shape. The spectroscopic studies confirm that all of these dyes can be degraded by laccase. Using MD simulations, we have demonstrated the presence of various distinct conformations of a loop in the protein active site that can accommodate the wide range of dye molecules. We have also shown that the diverse selection of dye molecules may exhibit surprisingly similar binding affinity due to cancellation of different thermodynamic factors. Our results highlight the potential of laccase as a multipurpose degrader for industrial dye effluents.

Phosphorylation-Competent Metastable State of Escherichia coli Toxin HipA.

Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA. Here we report a molten-globule-like intermediate of HipA at low urea (∼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in-out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be ∼2-2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.

Design of CO2 Thickeners and Role of Aromatic Rings in Enhanced Oil Recovery Using Molecular Dynamics.

Oligomers of PDMS (M1), polyFast (M2), modified PVEE (M3 and M4), and two new molecules with cyclic cores (M5 and M6) were studied to understand their ability to thicken the sc-CO2 at 377 K and 55 MPa, without any cosolvent. It was observed that PDMS and polyFast behaved in the known ways. PDMS does not improve the viscosity of the system without a cosolvent and PolyFast enhances the viscosity by a large margin. M3 and M4 also have not improved the viscosity significantly even with the introduction of a styrene component, but which has improved their solubilities in the fluid. M5 and M6, however, are observed to have enhanced the viscosity similar to that of polyFast due to their structural advantage and π-π interactions between the molecules. These molecules were also tested for their synthesizability, and their synthesis is found to be moderately easy.

Dimer-parity-dependent odd-even effects in photoinduced transitions to cholesteric and twist grain boundary smectic-C

We describe investigations on the influence of the flexible spacer parity and length of the guest photoactive liquid-crystalline dimers in guest-host mixtures exhibiting photoinduced transitions involving isotropic (I), cholesteric (N^{*}), and twist grain boundary smectic-C^{*} (TGBC^{*}) phases. Despite a small concentration (3 wt. %) of the guest molecules, the transition temperatures and their photodriven shift (δT) show a strong odd-even parity (of the dimer) dependent effect, with the even-parity systems having a larger value than their odd-parity counterparts; δT is larger for the N^{*}-TGBC^{*} transition than for the I-N^{*} one. The photocalorimetric measurements corroborate these features in addition to showing that, in comparison with the absence-of-ultraviolet (UV) case, the transition enthalpy (ΔH) of the I-N^{*} transition in the UV-on case is diminished by 33 and 12% for the mixtures with even- and odd-parity dimers, respectively. The duration for relaxation from the isothermal photodriven transition also exhibits general features of an odd-even influence. Molecular dynamics simulations demonstrate the presence of significant conformational heterogeneity and associated shift in the conformational space on photostimulation of the guest molecules. The change in the effective shape and nematic order parameter is more pronounced in the even-parity system.

Traceless cysteine-linchpin enables precision engineering of lysine in native proteins.

The maintenance of machinery requires its operational understanding and a toolbox for repair. The methods for the precision engineering of native proteins meet a similar requirement in biosystems. Its success hinges on the principles regulating chemical reactions with a protein. Here, we report a technology that delivers high-level control over reactivity, chemoselectivity, site-selectivity, modularity, dual-probe installation, and protein-selectivity. It utilizes cysteine-based chemoselective Linchpin-Directed site-selective Modification of lysine residue in a protein (LDMC-K). The efficiency of the end-user-friendly protocol is evident in quantitative conversions within an hour. A chemically orthogonal C-S bond-formation and bond-dissociation are essential among multiple regulatory attributes. The method offers protein selectivity by targeting a single lysine residue of a single protein in a complex biomolecular mixture. The protocol renders analytically pure single-site probe-engineered protein bioconjugate. Also, it provides access to homogeneous antibody conjugates (AFC and ADC). The LDMC-K-ADC exhibits highly selective anti-proliferative activity towards breast cancer cells.

Development of a P450 Fusion Enzyme for Biaryl Coupling in Yeast.

Despite the diverse and potent bioactivities displayed by axially chiral biaryl natural products, their application in drug discovery is limited by restricted access to these complex molecular scaffolds. In particular, fundamental challenges remain in controlling the site- and atroposelectivity in biaryl coupling reactions. In contrast, Nature has a wealth of biosynthetic enzymes that catalyze biaryl coupling reactions with catalyst-controlled selectivity. In particular, a growing subset of fungal P450s have been identified to catalyze site- and atroposelective biaryl couplings. Herein, we optimize a whole-cell biocatalytic platform in Pichia pastoris to synthesize biaryl molecules through the recombinant production of the fungal P450 KtnC. Moreover, engineering redox self-sufficient fusion enzymes further improves the efficiency of the system. Altogether, this work provides a platform for biaryl coupling reactions in yeast that can be applied to engineering a currently underexplored pool of fungal P450s into selective biocatalysts for the synthesis of complex biaryl compounds.

Biocatalytic oxidative cross-coupling reactions for biaryl bond formation.

Biaryl compounds, with two connected aromatic rings, are found across medicine, materials science and asymmetric catalysis1,2. The necessity of joining arene building blocks to access these valuable compounds has inspired several approaches for biaryl bond formation and challenged chemists to develop increasingly concise and robust methods for this task3. Oxidative coupling of two C-H bonds offers an efficient strategy for the formation of a biaryl C-C bond; however, fundamental challenges remain in controlling the reactivity and selectivity for uniting a given pair of substrates4,5. Biocatalytic oxidative cross-coupling reactions have the potential to overcome limitations inherent to numerous small-molecule-mediated methods by providing a paradigm with catalyst-controlled selectivity6. Here we disclose a strategy for biocatalytic cross-coupling through oxidative C-C bond formation using cytochrome P450 enzymes. We demonstrate the ability to catalyse cross-coupling reactions on a panel of phenolic substrates using natural P450 catalysts. Moreover, we engineer a P450 to possess the desired reactivity, site selectivity and atroposelectivity by transforming a low-yielding, unselective reaction into a highly efficient and selective process. This streamlined method for constructing sterically hindered biaryl bonds provides a programmable platform for assembling molecules with catalyst-controlled reactivity and selectivity.

Molecular Insight into Dye-Surfactant Interaction at Premicellar Concentrations: A Combined Two-Photon Absorption and Molecular Dynamics Simulation Study.

Both electrostatic and hydrophobic interactions play pivotal roles in ligand-surfactant binding interaction, especially for ionic surfactants. While much studies have been reported in the micellar region, less attention has been paid on such interactions at a low (premicellar) surfactant concentration. We here study the interaction between the cationic dye rhodamine 6G (R6G) with surfactants of different charge types: anionic SDS, cationic CTAB, and nonionic Tx 100 using absorption and emission spectroscopy. We identify that R6G forms dimeric aggregates at a premicellar concentration of SDS. Formation of aggregates is also confirmed from classical simulation measurements. CTAB and Tx 100 do not form any such aggregate, presumably owing to unfavorable electrostatic interactions. For a molecular-level understanding, we perform two-photon absorption (TPA) spectroscopy for the same systems. TPA allows us to calculate the two-photon absorption cross section and subsequently the change in the dipole moment (Δµ) between ground and excited states of the dye. We calculate the Δµ and observe that it passes through a maximum at a surfactant concentration half of the critical micelle concentration of SDS. This observation imparts support to earlier quantum mechanical calculation, which infers deviation from the parallel orientation of the dye during surfactant-induced aggregation. We extended our measurements and varied the carbon chain length of the anionic surfactant, and we found that all of them exhibit a maximum in Δµ, while their relative magnitude is dependent on the surfactant carbon chain length.

The Transformative Power of Biocatalysis in Convergent Synthesis.

Achieving convergent synthetic strategies has long been a gold standard in constructing complex molecular skeletons, allowing for the rapid generation of complexity in comparatively streamlined synthetic routes. Traditionally, biocatalysis has not played a prominent role in convergent laboratory synthesis, with the application of biocatalysts in convergent strategies primarily limited to the synthesis of chiral fragments. Although the use of enzymes to enable convergent synthetic approaches is relatively new and emerging, combining the efficiency of convergent transformations with the selectivity achievable through biocatalysis creates new opportunities for efficient synthetic strategies. This Perspective provides an overview of recent developments in biocatalytic strategies for convergent transformations and offers insights into the advantages of these methods compared to their small molecule-based counterparts.

How Far Is "Bulk Water" from Interfaces? Depends on the Nature of the Surface and What We Measure.

Using systematic molecular dynamics (MD) simulations, we revisit the question: At what distance from an interface do the properties of "bulk water" get recovered? We have considered three different kinds of interfaces: nonpolar (hydrophobic; isooctane-water interface), charged (negative; AOT bilayer), and polar (zwitterionic; POPC bilayer). In order to interrogate the extent of perturbation of the interfacial water molecules as a function of the distance from the interface, we utilize a diverse range of structural and dynamical parameters. To capture the structural perturbations, we look into local density (translational order), local tetrahedral order parameter, and dipolar orientation of the water molecules. We also explore the anisotropic diffusion of the water molecules in the direction perpendicular to the interface as well as the planar diffusion parallel to the interface in a distance dependent manner. In addition, the orientational time correlation functions have been computed to understand the extent of slowdown in the rotational dynamics. As expected, the electrostatic field emanating from the charged AOT interface seems to have the highest long-range effect on the orientational order and dynamics of the water molecules, whereas specific interactions like hydrogen bonding and electrostatic interaction lead to significant trapping and kinetic slowdown for both AOT and POPC (zwitterionic) very close to the interface. Our analysis highlights that not only the length-scale of perturbation depends on the nature of the interfaces and specific interactions but also the type of water property that we measure/calculate. Different water properties seem to have widely different length-scale of perturbation. Orientational order parameters seem to be perturbed to a much longer length-scale as compared to translational order parameters. The global orientational order of water can be perturbed even up to ∼4-5 nm near the negatively charged AOT surface in the absence of any extra electrolyte. This observation has significant implication toward the interpretation of experimental measurements as well since different spectroscopic techniques would probe different parameters or water properties with possible mutual disagreement and inconsistency between different types of measurements. Thus, our study provides a broader and unifying perspective toward the aspect of "context dependent" structural and dynamical perturbation of "interfacial water".

Size-Dependent Order-Disorder Crossover in Hydrophobic Hydration: Comparison between Spherical Solutes and Linear Alcohols.

Theory and computer simulation studies have predicted that water molecules around hydrophobic molecules should undergo an order-disorder transition with increasing solute size around a 1 nm length scale. Some theories predict the formation of a clathrate-like ordered structure around smaller hydrophobic solutes (<1 nm) and the formation of disordered vapor-liquid interfaces around larger solutes (>1 nm) and surfaces. Experimental validation of these predictions has often been elusive and contradictory. High-resolution Raman spectroscopy has detected that water around small hydrophobic solutes shows a signature similar to that of bulk water at lower temperature (increased ordering and a stronger hydrogen-bonded network). Similarly, water around larger solutes shows an increasing population of dangling OH bonds very similar to higher temperature bulk water. Thus, the solute size dependence of the structure and dynamics of water around hydrophobic molecules seems to have an analogy with the temperature dependence in bulk water. In this work, using atomistic classical molecular dynamics (MD) simulations, we have systematically investigated this aspect and characterized this interesting analogy. Structural order parameters including the tetrahedral order parameter (Q), hydrogen bond distribution, and vibrational power spectrum highlight this similarity. However, in contrast to the experimental observations, we do not observe any length-dependent crossover for linear hydrophobic alcohols (n-alkanols) using classical MD simulations. This is in agreement with earlier findings that linear alkane chains do not demonstrate the length-dependent order-disorder transition due to the presence of a sub-nanometer length scale along the cross section of the chain. Moreover, the collapsed state of linear hydrocarbon chains is not significantly populated for smaller chains (number of carbons below 20). In the context of our computational results, we raise several pertinent questions related to the sensitivity of various structural and dynamical parameters toward capturing these complex phenomena of hydrophobic hydration.

Prevalence and Covariates of Vitamin D Deficiencies (VDD) among Adolescents in India.

OBJECTIVE: To estimate the overall burden of vitamin D deficiencies (VDD) among Indian adolescents aged 10-19 y and to explore some selected covariates to determine a comprehensive guide to explore the vulnerable segments its guiding factors of VDD. METHODS: The study used secondary data of 11,822 adolescent children from the Community National Nutrition Survey (CNNS), undertaken in 2016-18. The data were analyzed by using STATA version 17.0. The multinomial logistic regression model was used to explore the covariates of VDD after adjusting all multicollinearity and giving analytical as well as sampling weights. RESULTS: The results revealed that nearly one-fourth of adolescent children have VDD (23.46%, 95% CI: 22.69%-24.22%) across India. Children belonging to the Hindu caste population, children who occasionally (and not weekly), or never, consume eggs, children living in north Indian states specifically in Punjab, Haryana, and Uttarakhand, children belonging to the richest households (wealth index-wise), and children suffering from overweight and obesity were more inclined to VDD. In the final adjusted multinomial regression model, the odds of VDD were significantly higher among urban living children. CONCLUSIONS: Findings concluded that proper intervention programs targeting specific population groups and/or regions of India are essential to combat the burden of VDD for enriching India's sustainable development goal of eradicating hunger by 2030.

State-of-the-Art Biocatalysis.

The use of enzyme-mediated reactions has transcended ancient food production to the laboratory synthesis of complex molecules. This evolution has been accelerated by developments in sequencing and DNA synthesis technology, bioinformatic and protein engineering tools, and the increasingly interdisciplinary nature of scientific research. Biocatalysis has become an indispensable tool applied in academic and industrial spheres, enabling synthetic strategies that leverage the exquisite selectivity of enzymes to access target molecules. In this Outlook, we outline the technological advances that have led to the field's current state. Integration of biocatalysis into mainstream synthetic chemistry hinges on increased access to well-characterized enzymes and the permeation of biocatalysis into retrosynthetic logic. Ultimately, we anticipate that biocatalysis is poised to enable the synthesis of increasingly complex molecules at new levels of efficiency and throughput.

Structural and dynamical heterogeneity of water trapped inside Na+-pumping KR2 rhodopsin in the dark state.

Photoisomerization in the retinal leads to a channel opening in rhodopsins that triggers translocation or pumping of ions/protons. Crystal structures of rhodopsins contain several structurally conserved water molecules. It has been suggested that water plays an active role in facilitating the ion pumping/translocation process by acting as a lubricant in these systems. In this paper, we systematically investigate the localization, structure, dynamics, and energetics of the water molecules along the channel for the resting/dark state of KR2 rhodopsin. By employing several microseconds long atomistic molecular dynamics simulation of this trans-membrane protein system, we demonstrate the presence of five distinct water containing pockets/cavities separated by gateways controlled by protein side-chains. There exists a strong hydrogen bonded network involving these buried water molecules and functionally important key residues. We present evidence of significant structural and dynamical heterogeneity in the water molecules present in these cavities, with very rare exchange between them. The exchange time scale of such buried water with the bulk has an extremely wide range, from tens of nanoseconds to >1.5 µs. The translational and rotational dynamics of buried water are found to be strongly dependent on the protein cavity size and local interactions with a classic signature of trapped diffusion and rotational anisotropy.