Loop Fluctuations Research Articles

Molecular Mechanism of Ciprofloxacin Translocation Through the Major Diffusion Channels of the ESKAPE Pathogens Klebsiella pneumoniae and Enterobacter cloacae.

Experimental studies on the translocation and accumulation of antibiotics in Gram-negative bacteria have revealed details of the properties that allow efficient permeation through bacterial outer membrane porins. Among the major outer membrane diffusion channels, OmpF has been extensively studied to understand the antibiotic translocation process. In a few cases, this knowledge has also helped to improve the efficacy of existing antibacterial molecules. However, the extension of these strategies to enhance the efficacy of other existing and novel drugs require comprehensive molecular insight into the permeation process and an understanding of how antibiotic and channel properties influence the effective permeation rates. Previous studies have investigated how differences in antibiotic charge distribution can influence the observed permeation pathways through the OmpF channel, and have shown that the dynamics of the L3 loop can play a dominant role in the permeation process. Here, we perform all-atom simulations of the OmpF orthologs, OmpE35 from Enterobacter cloacae and OmpK35 from Klebsiella pneumoniae. Unbiased simulations of the porins and biased simulations of the ciprofloxacin permeation processes through these channels provide insight into the differences in the permeation pathway and energetics. In addition, we show that similar to the OmpF channel, antibiotic-induced dynamics of the L3 loop are also operative in the orthologs. However, the sequence and structural differences, influence the extent of the L3 loop fluctuations with OmpK35 showing greater stability in unbiased runs and subdued fluctuations in simulations with ciprofloxacin.

RNA's Dynamic Conformational Selection and Entropic Allosteric Mechanism in Controlling Cascade Protein Binding Events.

In the TAR RNA of immunodeficiency viruses, an allosteric communication exists between a distant loop and a bulge. The bulge interacts with the TAT protein vital for transactivating viral RNA, while the loop interacts with cyclin-T1, contingent on TAT binding. Through extensive atomistic and free energy simulations, we investigate TAR-TAT binding in nonpathogenic bovine immunodeficiency virus (BIV) and pathogenic human immunodeficiency virus (HIV). Thermodynamic analysis reveals enthalpically driven binding in BIV and entropically favored binding in HIV. The broader global basin in HIV is attributed to binding-induced loop fluctuation, corroborated by nuclear magnetic resonance (NMR), indicating classical entropic allostery onset. While this loop fluctuation affects the TAT binding affinity, it generates a binding-competent conformation that aids subsequent effector (cyclin-T1) binding. This study underscores how two structurally similar apo-RNA scaffolds adopt distinct conformational selection mechanisms to drive enthalpic and entropic allostery, influencing protein affinity in the signaling cascade.

Cryo-EM structures reveal two allosteric inhibition modes of PI3KαH1047R involving a re-shaping of the activation loop

PI3Kα is a lipid kinase that phosphorylates PIP2 and generates PIP3. The hyperactive PI3Kα mutation, H1047R, accounts for about 14% of breast cancer, making it a highly attractive target for drug discovery. Here, we report the cryo-EM structures of PI3KαH1047R bound to two different allosteric inhibitors QR-7909 and QR-8557 at a global resolution of 2.7Å and 3.0Å, respectively. The structures reveal two distinct binding pockets on the opposite sides of the activation loop. Structural and MD simulation analyses show that the allosteric binding of QR-7909 and QR-8557 inhibit PI3KαH1047R hyper-activity by reducing the fluctuation and mobility of the activation loop. Our work provides a strong rational basis for a further optimization and development of highly selective drug candidates to treat PI3KαH1047R-driven cancers.

Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation

BackgroundLignocellulose is the most abundant natural biomass resource for the production of biofuels and other chemicals. The efficient degradation of cellulose by cellulases is a critical step for the lignocellulose bioconversion. Understanding the structure-catalysis relationship is vital for rational design of more stable and highly active enzymes. Glycoside hydrolase (GH) family 5 is the largest and most functionally diverse group of cellulases, with a conserved TIM barrel structure. The important roles of the various loop regions of GH5 enzymes in catalysis, however, remain poorly understood.ResultsIn the present study, we investigated the relationship between the loops surrounding active site architecture and its catalytic efficiency, taking TfCel5A, an enzyme from GH5_2 subfamily of Thermobifida fusca, as an example. Large-scale computational simulations and site-directed mutagenesis experiments revealed that three loops (loop 8, 3, and 7) around active cleft played diverse roles in substrate binding, intermediate formation, and product release, respectively. The highly flexible and charged residue triad of loop 8 was responsible for capturing the ligand into the active cleft. Severe fluctuation of loop 3 led to the distortion of sugar conformation at the − 1 subsite. The wobble of loop 7 might facilitate product release, and the enzyme activity of the mutant Y361W in loop 7 was increased by approximately 40%.ConclusionThis study unraveled the vital roles of loops in active site architecture and provided new insights into the catalytic mechanism of the GH5_2 cellulases.

Bioactivity-Guided Synthesis: In Silico and In Vitro Studies of β-Glucosidase Inhibitors to Cope with Hepatic Cytotoxicity.

The major cause of hyperglycemia can generally be attributed to β-glucosidase as per its involvement in non-alcoholic fatty liver disease. This clinical condition leads to liver carcinoma (HepG2 cancer). The phthalimides and phthalamic acid classes possess inhibitory potential against glucosidase, forming the basis for designing new phthalimide and phthalamic acid analogs to test their ability as potent inhibitors of β-glucosidase. The study also covers in silico (molecular docking and MD simulations) and in vitro (β-glucosidase and HepG2 cancer cell line assays) analyses. The phthalimide and phthalamic acid derivatives were synthesized, followed by spectroscopic characterization. The mechanistic complexities associated with β-glucosidase inhibition were identified via the docking of the synthesized compounds inside the active site of the protein, and the results were analyzed in terms of the best binding energy and appropriate docking pose. The top-ranked compounds were subjected to extensive MD simulation studies to understand the mode of interaction of the synthesized compounds and binding energies, as well as the contribution of individual residues towards binding affinities. Lower RMSD/RMSF values were observed for 2c and 3c, respectively, in the active site, confirming more stabilized, ligand-bound complexes when compared to the free state. An anisotropic network model was used to unravel the role of loop fluctuation in the context of ligand binding and the dynamics that are distinct to the bound and free states, supported by a 3D surface plot. An in vitro study revealed that 1c (IC50 = 1.26 µM) is far better than standard acarbose (2.15 µM), confirming the potential of this compound against the target protein. Given the appreciable potential of the candidate compounds against β-glucosidase, the synthesized compounds were further tested for their cytotoxic activity against hepatic carcinoma on HepG2 cancer cell lines. The cytotoxicity profile of the synthesized compounds was performed against HepG2 cancer cell lines. The resultant IC50 value (0.048 µM) for 3c is better than the standard (thalidomide: IC50 0.053 µM). The results promise the hypothesis that the synthesized compounds might become potential drug candidates, given the fact that the β-glucosidase inhibition of 1c is 40% better than the standard, whereas compound 3c holds more anti-tumor activity (greater than 9%) against the HepG2 cell line than the known drug.

Reconstruction of the unbinding pathways of new inhibitors of the SARS-CoV-2 papain-like protease using molecular dynamics simulation

Developing novel antiviral drugs against the SARS-CoV-2 virus and COVID-19 disease is imperative as the vaccines may not offer absolute protection. PLpro plays a crucial role in the viral life cycle, making it an attractive target for drug development. Several PLpro inhibitors have been developed, and their 3D structures in complex with PLpro are available. In this work, we employed Supervised Molecular Dynamics (SuMD), a specific Unbiased Molecular Dynamics (UMD) method, to investigate unbinding pathways of the novel inhibitors of PLpro (PDB IDs: 7LBR, 7RZC, 7SDR and 7E35) and GRL0617 (PDB ID: 7JRN) as a reference. We conducted three simulations for each ligand and achieved unbinding events in the nanosecond timescale in all simulations. We found that unbinding events are commonly affected by altering the conformation of the BL2 loop, which is caused by the natural fluctuations of the loop that are required to trap the substrate and throw out the product. BL2 loop is crucial for keeping the ligand and unbinding and acts as a double-edged sword. Any inhibitor designed to be effective must prevent the loop’s natural fluctuations. We perceived that increasing ligands interactions with the binding pocket interior and the BL2 loop will help prevent natural fluctuation of the BL2 loop, Although the interactions with the binding pocket’s inner side are more critical than the BL2 loop. These findings may be helpful in developing more potent inhibitors against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

Topological Fracton Quantum Phase Transitions by Tuning Exact Tensor Network States.

Gapped fracton phases of matter generalize the concept of topological order and broaden our fundamental understanding of entanglement in quantum many-body systems. However, their analytical or numerical description beyond exactly solvable models remains a formidable challenge. Here we employ an exact 3D quantum tensor-network approach that allows us to study a Z_{N} generalization of the prototypical X cube fracton model and its quantum phase transitions between distinct topological states via fully tractable wave function deformations. We map the (deformed) quantum states exactly to a combination of a classical lattice gauge theory and a plaquette clock model, and employ numerical techniques to calculate various entanglement order parameters. For the Z_{N} model we find a family of (weakly) first-order fracton confinement transitions that in the limit of N→∞ converge to a continuous phase transition beyond the Landau-Ginzburg-Wilson paradigm. We also discover a line of 3D conformal quantum critical points (with critical magnetic flux loop fluctuations) which, in the N→∞ limit, appears to coexist with a gapless deconfined fracton state.

A Single-Event-Hardened Scheme of Phase-Locked Loop Microsystems for Aerospace Applications.

In order to improve the ability of the phase-locked loop (PLL) microsystem applied in the aerospace environment to suppress the irradiation effect, this study presents an efficient charge pump hardened scheme by using the radiation-hardened-by-design (RHBD) technology. In this study, the sensitivity analysis of the single-event transient (SET) at different nodes of charge pump and different bombardment energies is carried out. Without changing the original structure and loop parameters, a hardened scheme of phase-locked loop to suppress the single-event effect is proposed. A digital control circuit is added between the charge pump and low-pass filter, which greatly reduces the sensitivity of the charge pump to the SET. The classical double-exponential current pulse model is used to simulate the SET effect on the unreinforced and reinforced phase-locked loops, and the reliability of the proposed reinforcement scheme is verified. The simulation results based on the SMIC 130 nm standard complementary metal-oxide-semiconductor (CMOS) process show that the peak value of the transient response fluctuation of the phase-locked loop using the proposed single-event-hardened scheme decreased by 94.2%, the lock recovery time increased by 75.3%, and the maximum phase shift decreased by 90.8%. This shows that the hardened scheme can effectively reduce the sensitivity of the PLL microsystems to the SET effects.

Rational engineering of phospholipase C from Bacillus cereus HSL3 for simultaneous thermostability and activity improvement

As an essential enzyme for phospholipid degradation, phospholipase C (PLC) has been used for enzymatic degumming of vegetable oils and production of valuable phospholipid derivatives. In this study, rational engineering based on B-factor analysis and molecular dynamic simulation analysis were employed to rationally identify mutation candidates and a PLC double mutant F96R/Q153P was designed from Bacillus cereus HSL3. Compared to the wild-type PLC, F96R/Q153P exhibited significantly improved thermal properties, including higher temperature optima and better thermal stability. It showed the highest optimal reaction temperature (90 °C) reported so far. F96R/Q153P displayed 4.94 times kcat and 2.37 times kcat/Km as much as the wild-type, as well as improved substrate adaptability. Structural insights revealed that the mutations caused reduced proportion of random coil and constraint of certain loop fluctuations. These results demonstrated the great potential of knowledge-based rational design for improving the catalytic characteristics of industrial enzymes in the enzymatic degumming process.

Correlating confinement to topological fluctuations near the crossover transition in QCD

We show the existence of strong (anti)correlations between the topological hot spots and the local values of the trace of the Polyakov loop in $2+1$ flavor QCD with physical quark mass, in the vicinity of the crossover transition corresponding to the simultaneous restoration of chiral symmetry and deconfinement. Using sophisticated lattice techniques, we have carefully identified the topological hot spots using quark zero modes and measured the short-distance fluctuations of the Polyakov loop about them, showing how the latter is repelled quite strongly around the peak of the zero modes. Though we could explain some aspects of these correlations within the instanton-dyon picture, our work sets the stage for a larger goal towards a systematic study of the role of different topological species that interact with the Polyakov loop, establishing the strong connection between topology and confinement.

Effects of Integrated Fuzzy Logic PID Controller on Satellite Antenna Tracking System.

An electrical device that transforms the electricity into the waves of radio and vice versa is termed the antenna. Its main deployment is in the transmitter and receiver of the antenna. While transmission, the transmitter of radio at the extremities of the antenna furnishes the electricity which oscillates at the frequency of radio wave and energy is released as current as em waves. Some of the voltage is formed from the em wave that is invaded at the point of receiving to amplify the receiver. This study focuses on the analysis of the satellite system to aid in mobile antenna tracking. It also examines the techniques for fuzzy control which make up traditional networks that are used. Initially, a basic idea of tracking loops with stabilized antennas was suggested in light of the requirement for the margin of phase and bandwidth. If the gain of the track is reduced due to changes in attributes and throughput, it will be reduced. In addition, fuzzy regulators and PID constituents are used to enhance the loop. The results indicate that the higher and lower antenna tracking gains within the loop were the best fit and the loop's fluctuations are reduced. A controller based on fuzzy logic can be most efficient due to its simplicity and robustness. It is also discovered that fuzzy logic controllers are evaluated by their behavior in relation. This paper presents an evaluation of the controllers in fuzzy logic, which is based on its integration with conventional controllers. There are three gains in PID's regulator PID and every gain can be used to control the variables of inputs and outcomes. The effects of the responses were analyzed and were compared. The commonality was discovered in the results according to the increase in time for II/6 and II/3 based on PID's regulator PID stability, it can be improved by this system, and there is a reduction in the duration of stability. Furthermore, the period of stability may be reduced through the fusion of PID and fuzzy. The effectiveness of the system could be enhanced by the implementation of the neural network. It is also possible to design the two types of control that could be used to control the proposed solid platform.

Localization of Dirac modes in finite-temperature Z2 gauge theory on the lattice

We study the localization properties of the eigenmodes of the staggered Dirac operator in finite-temperature $\mathbb{Z}_2$ pure gauge theory on the lattice in 2+1 dimensions. We find that the low modes turn from delocalized to localized as the system crosses over from the confined to the deconfined phase in the "physical" sector (positive average Polyakov loop) selected by external fermionic probes, while they remain delocalized in the "unphysical" sector (negative average Polyakov loop). This confirms that the close connection between deconfinement and localization of the low Dirac modes in the physical sector, already observed in other models, holds also in the simplest gauge theory displaying a deconfinement transition. We also observe a clear correlation of localized modes with fluctuations of the Polyakov loop away from the ordered value, as expected according to the "sea/islands" picture of localization, and with clusters of negative plaquettes. A novel finding is the presence of localized modes at the high end of the Dirac spectrum in all phases/sectors of the theory.

Bridging the Bridging Imidazolate in the Bimetallic Center of the Cu/Zn SOD1 and ALS.

Metallation status of human Cu/Zn superoxide dismutase 1 (SOD1) plays a pivotal role in the pathogenesis of amyotrophic lateral sclerosis (ALS). All of the amino acids found in the bimetallic center have been associated with ALS except for two positions. H63 which forms the bridging imidazolate ion in the bimetallic center and K136 which is not directly involved in coordination but located in the bimetallic center were not reported to be mutated in any of the identified ALS cases. In this study, we investigated the structure and flexibility of five SOD1 variants by using classical molecular dynamics simulations. These variants include three substitutions on the non-ALS-linked positions; H63A, H63R, K136A and ALS-linked positions; G37R, H46R/H48D. We have generated four systems for each variant differing in metallation and presence of the intramolecular disulfide bond. Overall, a total of 24 different dimers including the wild-type were generated and simulated at two temperatures, 298 and 400 K. We have monitored backbone mobility, fluctuations and compactness of the dimer structures to assess whether the hypothetical mutations would behave similar to the ALS-linked variants. Results showed that particularly two mutants, H63R and K136A, drastically affected the dimer dynamics by increasing the fluctuations of the metal binding loops compared with the control mutations. Further, these variants resulted in demetallation of the dimers, highlighting probable ALS toxicity that could be elicited by the SOD1 variants of H63R and K136A. Overall, this study bridges two putative SOD1 positions in the metallic center and ALS, underlining the potential use of atomistic simulations for studying disease variants.

Structures and topological defects in pressure-driven lyotropic chromonic liquid crystals

Lyotropic chromonic liquid crystals are water-based materials composed of self-assembled cylindrical aggregates. Their behavior under flow is poorly understood, and quantitatively resolving the optical retardance of the flowing liquid crystal has so far been limited by the imaging speed of current polarization-resolved imaging techniques. Here, we employ a single-shot quantitative polarization imaging method, termed polarized shearing interference microscopy, to quantify the spatial distribution and the dynamics of the structures emerging in nematic disodium cromoglycate solutions in a microfluidic channel. We show that pure-twist disclination loops nucleate in the bulk flow over a range of shear rates. These loops are elongated in the flow direction and exhibit a constant aspect ratio that is governed by the nonnegligible splay-bend anisotropy at the loop boundary. The size of the loops is set by the balance between nucleation forces and annihilation forces acting on the disclination. The fluctuations of the pure-twist disclination loops reflect the tumbling character of nematic disodium cromoglycate. Our study, including experiment, simulation, and scaling analysis, provides a comprehensive understanding of the structure and dynamics of pressure-driven lyotropic chromonic liquid crystals and might open new routes for using these materials to control assembly and flow of biological systems or particles in microfluidic devices.

Correlation of membrane protein conformational and functional dynamics

Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

In silico screening and analysis of nonsynonymous SNPs in human CYP1A2 to assess possible associations with pathogenicity and cancer susceptibility

Cytochrome P450 1A2 (CYP1A2) is one of the main hepatic CYPs involved in metabolism of carcinogens and clinically used drugs. Nonsynonymous single nucleotide polymorphisms (nsSNPs) of this enzyme could affect cancer susceptibility and drug efficiency. Hence, identification of human CYP1A2 pathogenic nsSNPs could be of great importance in personalized medicine and pharmacogenetics. Here, 176 nsSNPs of human CYP1A2 were evaluated using a variety of computational tools, of which 18 nsSNPs were found to be associated with pathogenicity. Further analysis suggested possible association of 9 nsSNPs (G73R, G73W, R108Q, R108W, E168K, E346K, R431W, F432S and R456H) with the risk of hepatocellular carcinoma. Molecular dynamics simulations revealed higher overall flexibility, decreased intramolecular hydrogen bonds and lower content of regular secondary structures for both cancer driver variants G73W and F432S when compared to the wild-type structure. In case of F432S, loss of the conserved hydrogen bond between Arg137 and heme propionate oxygen may affect heme stability and the observed significant rise in fluctuation of the CD loop could modify CYP1A2 interactions with its redox partners. Together, these findings propose CYP1A2 as a possible candidate for hepatocellular carcinoma and provide structural insights into how cancer driver nsSNPs could affect protein structure, heme stability and interaction network.

Terahertz physical mechanism of arsenic (III) restoring p53 mutant protein activity

Recovering the protein activity of p53 mutants through small molecule ligand binding (eg. arsenic) is an important strategy for tumor suppressor therapy. However, the mechanistic basis on the changes of collective dynamics and their roles of p53 protein in functional recovery process has not been fully elucidated. Herein, the normal mode calculations based on all-atom elastic network model are employed to characterize the terahertz low frequency motions of core DNA-binding domain (p53C) which is essential for p53 protein activities in transcriptional transactivation. We find that the lowest-frequency collective vibration mode of the p53C mutant is effectively restored by the binding of arsenic (III) ligand. In R249S mutant, the L1 loop is stabilized through restricting the swing-out movement. The results obtained from atomic backbone fluctuations suggest that the arsenic binding can significantly improve the L1 loop and L2 loop fluctuations. The statistical analysis of low frequency vibration mode reflects that the arsenic-bound R249S mutant has an apparent recovery of frequency shift in the terahertz range. The residue-residue motion correlation also suggests that structural components binding to arsenic are dynamically coupled. In the H2 helix with arsenic-binding residues, the motions of C124, C135, M133 and C141, are correlated with the arsenic recovery. These results provide the terahertz biophysical mechanism for the recovery effect of arsenic (III) on the p53 protein activity and new evidence for the coupling of the low-frequency vibration characteristics of protein structures with its function, thus giving a new physical insight into the p53 related cancer therapies.

Steady-state fluctuations of a genetic feedback loop with fluctuating rate parameters using the unified colored noise approximation

A common model of stochastic auto-regulatory gene expression describes promoter switching via cooperative protein binding, effective protein production in the active state and dilution of proteins. Here we consider an extension of this model whereby colored noise with a short correlation time is added to the reaction rate parameters—we show that when the size and timescale of the noise is appropriately chosen it accounts for fast reactions that are not explicitly modeled, e.g., in models with no mRNA description, fluctuations in the protein production rate can account for rapid multiple stages of nuclear mRNA processing which precede translation in eukaryotes. We show how the unified colored noise approximation can be used to derive expressions for the protein number distribution that is in good agreement with stochastic simulations. We find that even when the noise in the rate parameters is small, the protein distributions predicted by our model can be significantly different than models assuming constant reaction rates.

Deconfinement in the presence of a strong magnetic field

We study the impact of a finite magnetic field on the deconfinement phase transition for heavy quarks by computing the fluctuations of the Polyakov loops. It is demonstrated that the explicit Z(3) breaking field increases with the magnetic field, leading to a decrease in the (pseudo) critical temperatures and a shrinking first-order region in the phase diagram. Phenomenological equations that capture the behaviors of the Z(3) breaking field at strong and weak magnetic fields for massive and massless quarks are given. Lastly, we explore the case of dynamical light quarks and demonstrate how an improved constituent quark mass function can enforce the correct magnetic field dependence of the deconfinement temperature in an effective model, as observed in Lattice QCD calculations.

Enhancement of keratin-degradation ability of the keratinase KerBL from Bacillus licheniformis WHU by proximity-triggered chemical crosslinking

Keratinases are valuable enzymes, given their application in keratin-rich waste recycling. Considering that keratinases usually require reducing agents to efficiently degrade keratin, improving the stability of keratinases under reducing conditions is highly desirable for practical applications. Here, we show that the introduction of several tyrosine derivatives containing para-substituted long-chain haloalkanes into the keratinase KerBL, which enabled proximity-triggered covalent crosslinking by rational design, could improve both the thermostability and autolytic resistance of the enzyme. After screening a series of noncanonical amino acid (ncAA)-based variants generated by rational design, two variants, N159C/Y260BprY and N159C/Y260BbtY, with enhanced keratinolytic activity were obtained. Both variants increased the Tm of the enzyme by approximately 10 °C. The potential mechanism underlying these improvements was investigated by molecular dynamics (MD) analysis. The results indicated that BprY-Cys and BbtY-Cys covalent bonds in the N159C/Y260TAG variant could significantly decrease the flexibility and fluctuations of the long loop (residues 151–162).