Biophysical Approaches Research Articles

A computational model to uncover the biophysical underpinnings of neural firing heterogeneity in dissociated hippocampal cultures.

Calcium (Ca2+ ) imaging reveals a variety of correlated firing in cultures of dissociated hippocampal neurons, pinpointing the non-synaptic paracrine release of glutamate as a possible mediator for such firing patterns, although the biophysical underpinnings remain unknown. An intriguing possibility is that extracellular glutamate could bind metabotropic receptors linked with inositol trisphosphate (IP3 ) mediated release of Ca2+ from the endoplasmic reticulum of individual neurons, thereby modulating neural activity in combination with sarco/endoplasmic reticulum Ca2+ transport ATPase (SERCA) and voltage-gated Ca2+ channels (VGCC). However, the possibility that such release may occur in different neuronal compartments and can be inherently stochastic poses challenges in the characterization of such interplay between various Ca2+ channels. Here we deploy biophysical modeling in association with Monte Carlo parameter sampling to characterize such interplay and successfully predict experimentally observed Ca2+ patterns. The results show that the neurotransmitter level at the plasma membrane is the extrinsic source of heterogeneity in somatic Ca2+ transients. Our analysis, in particular, identifies the origin of such heterogeneity to an intrinsic differentiation of hippocampal neurons in terms of multiple cellular properties pertaining to intracellular Ca2+ signaling, such as VGCC, IP3 receptor, and SERCA expression. In the future, the biophysical model and parameter estimation approach used in this study can be upgraded to predict the response of a system of interconnected neurons.

Clp1, an overexpressed subunit of CF IA and insights into its molecular role using biophysical approaches

In eukaryotes, pre-messenger RNA (pre-mRNA) 3′-end processing is vital for gene expression and mRNA biogenesis; any awry in this pre-mRNA processing has detrimental effects and causes various diseases in humans. Pre-mRNA processing is coordinated via complex multi-protein machinery involving over 20 protein factors in Saccharomyces cerevisiae. Endonucleolytic cleavage at the pre-mRNA 3′-end followed by poly(A) tail addition at the newly generated 3′-end requires CF IA, CPF, CF IB and other sequence elements. Among these essential proteins, Clp1 of the CF IA sub-unit is indispensable for the effective functioning of 3′-end processing machinery. Clp1, a conserved protein, is proposed to interlink 3′-end processing, transcriptional elongation and transcription termination. However, experimental evidence defining the precise roles played by Clp1, its interacting partners from other subunits and information about its exact mechanism of action during pre-mRNA processing is lacking. The current study details on the Clp1 N-terminal domain's (1–100 amino acids referred to as Clp11–100) successful cloning, expression and characterization using numerous biophysical techniques like circular dichroism and mass spectrometry etc. This information would open new frontiers to decipher the structure and molecular role of Clp1 during pre-mRNA maturation.

Targeting spike glycans to inhibit SARS-CoV2 viral entry

SARS-CoV-2 spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical approaches, we demonstrate that the interaction is avidity driven and that BOA cross-links the spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that multivalent glycan-targeting molecules have the potential to act as pan-coronavirus inhibitors.

Unraveling the unexpected aggregation behavior of Pyrazole-Based compounds Targeting Mycobacterium tuberculosis UDP-Galactopyranose mutase

A pyrazole-based compound, MS208, was previously identified as an inhibitor of UDP-Galactopyranose Mutase from Mycobacterium tuberculosis (MtUGM). Targeting this enzyme is a novel therapeutic strategy for the development of new antituberculosis agents because MtUGM is an essential enzyme for the bacterial cell wall synthesis and it is not present in human. It was proposed that MS208 targets an allosteric site in MtUGM as MS208 followed a mixed inhibition model. DA10, an MS208 analogue, showed competitive inhibition rather than mixed inhibition. In this paper, we have used an integrated biophysical approach, including thermal shift assays, dynamic light scattering and nuclear magnetic resonance experiments, to show that MS208 and many analogues displayed unexpected aggregation behavior against MtUGM.

Inhibition of β-lactamase function by de novo designed peptide.

Antimicrobial resistance is a great public health concern that is now described as a "silent pandemic". The global burden of antimicrobial resistance requires new antibacterial treatments, especially for the most challenging multidrug-resistant bacteria. There are various mechanisms by which bacteria develop antimicrobial resistance including expression of β-lactamase enzymes, overexpression of efflux pumps, reduced cell permeability through downregulation of porins required for β-lactam entry, or modifications in penicillin-binding proteins. Inactivation of the β-lactam antibiotics by β-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Although several effective small-molecule inhibitors of β-lactamases such as clavulanic acid and avibactam are clinically available, they act only on selected class A, C, and some class D enzymes. Currently, none of the clinically approved inhibitors can effectively inhibit Class B metallo-β-lactamases. Additionally, there is increased resistance to these inhibitors reported in several bacteria. The objective of this study is to use the Resonant Recognition Model (RRM), as a novel strategy to inhibit/modulate specific antimicrobial resistance targets. The RRM is a bio-physical approach that analyzes the distribution of energies of free electrons and posits that there is a significant correlation between the spectra of this energy distribution and related protein biological activity. In this study, we have used the RRM concept to evaluate the structure-function properties of a group of 22 β-lactamase proteins and designed 30-mer peptides with the desired RRM spectral periodicities (frequencies) to function as β-lactamase inhibitors. In contrast to the controls, our results indicate 100% inhibition of the class A β-lactamases from Escherichia coli and Enterobacter cloacae. Taken together, the RRM model can likely be utilized as a promising approach to design β-lactamase inhibitors for any specific class. This may open a new direction to combat antimicrobial resistance.

Biophysical Insights into Drug Discovery: Leveraging Phase Transitions and Protein Behavior for Therapeutic Innovation

In the search for novel treatments for various diseases, the nexus of biophysics and drug discovery represents a dynamic and transformational paradigm. A new age in pharmaceutical research has begun thanks to biophysics and its thorough grasp of the structural and physical characteristics of biological molecules. This study explores how advances in biophysical approaches, including allosteric modulation, intrinsically disordered proteins (IDPs), and protein phase transitions, have transformed the drug development process. Protein phase transitions, supported by the biophysical principles, have provided crucial insights into disorders like ALS and Alzheimer’s, opening up new avenues for therapeutic intervention. Targeting IDPs and their role in liquid–liquid phase separation has produced creative approaches to diseases that were formerly thought to be resistant to therapy. In order to reduce the complexity of complicated diseases, the concept of allosteric modulation, made possible by biophysical understanding, offers a precise and selective approach to medication creation. This review highlights the significant influence of biophysics on the development of therapeutics and the discovery of new drugs. Biophysics is advancing the discipline toward the creation of more precise, efficient, and tailored medications by illuminating the biophysical properties of proteins, the complexities of phase transitions, and the dynamics of drug interactions. Biophysical insights are poised to alter healthcare by bringing together several fields and sustained innovation, giving people dealing with a variety of ailments fresh hope.

Increased Actin Binding Is a Shared Molecular Consequence of Numerous SCA5 Mutations in β-III-Spectrin.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein β-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the β-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with an early age of symptom onset. Altogether, the data indicate that increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.

Disruption of Ebola NP0VP35 Inclusion Body-like Structures reduce Viral Infection

Viral inclusion bodies (IBs) are potential sites of viral replication and assembly. How viral IBs form remains poorly defined. Here we describe a combined biophysical and cellular approach to identify the components necessary for IB formation during Ebola virus (EBOV) infection. We find that the eNP0VP35 complex containing Ebola nucleoprotein (eNP) and viral protein 35 (eVP35), the functional equivalents of nucleoprotein (N) and phosphoprotein (P) in non-segmented negative strand viruses (NNSVs), phase separates to form inclusion bodies. Phase separation of eNP0VP35 is reversible and modulated by ionic strength. The multivalency of eVP35, and not eNP, is also critical for phase separation. Furthermore, overexpression of an eVP35 peptide disrupts eNP0VP35 complex formation, leading to reduced frequency of IB formation and limited viral infection. Together, our results show that upon EBOV infection, the eNP0VP35 complex forms the minimum unit to drive IB formation and viral replication.

Lipid clearance and amyloid formation by serum amyloid A: exploring the links between beneficial and pathologic actions of an enigmatic protein

Serum amyloid A (SAA) is named after a life-threatening disease, yet this small evolutionarily conserved protein must have played a vital role in host defense. Most circulating SAA binds plasma lipoproteins and modulates their metabolism. However, this hardly justifies the rapid and dramatic SAA upregulation in inflammation, which is concomitant with upregulation of secretory phospholipase A2 (sPLA2). We proposed that these proteins synergistically clear cell membrane debris from the sites of injury. The present study uses biochemical and biophysical approaches to further explore the beneficial function of SAA and its potential links to amyloid formation. We show that murine and human SAA1 are powerful detergents that solubilize diverse lipids, including mammalian biomembranes, converting them into lipoprotein-size nanoparticles. These nanoparticles provide ligands for cell receptors, such as scavenger receptor CD36 or heparin/heparan sulfate, act as substrates of sPLA2, and sequester toxic products of sPLA2. Together, these functions enable SAA to rapidly clear unprotected lipids. SAA can also adsorb, without remodeling, to lipoprotein-size nanoparticles such as exosomal liposomes, which are proxies for lipoproteins. SAA in complexes with zwitterionic phospholipids stabilizes α-helices, while SAA in complexes containing anionic lipids or micelle-forming sPLA2 products forms metastable β-sheet–rich species that readily aggregate to form amyloid. Consequently, the synergy between SAA and sPLA2 extends from the beneficial lipid clearance to the pathologic amyloid formation. Furthermore, we show that lipid composition alters SAA conformation and thereby can influence the metabolic fate of SAA–lipid complexes, including their proamyloidogenic and proatherogenic binding to heparan sulfate.

Aldosterone Antagonism Is More Effective at Reducing Blood Pressure and Excessive Renal ENaC Activity in AngII-Infused Female Rats Than in Males.

AngII (angiotensin II)-dependent hypertension causes comparable elevations of blood pressure (BP), aldosterone levels, and renal ENaC (epithelial Na+ channel) activity in male and female rodents. Mineralocorticoid receptor (MR) antagonism has a limited antihypertensive effect associated with insufficient suppression of renal ENaC in male rodents with AngII-hypertension. While MR blockade effectively reduces BP in female mice with salt-sensitive and leptin-induced hypertension, MR antagonism has not been studied in female rodents with AngII-hypertension. We hypothesize that overstimulation of renal MR signaling drives redundant ENaC-mediated Na+ reabsorption and BP increase in female rats with AngII-hypertension. We employ a combination of physiological, pharmacological, biochemical, and biophysical approaches to compare the effect of MR inhibitors on BP and ENaC activity in AngII-infused male and female Sprague Dawley rats. MR blockade markedly attenuates AngII-hypertension in female rats but has only a marginal effect in males. Spironolactone increases urinary sodium excretion and urinary sodium-to-potassium ratio in AngII-infused female, but not male, rats. The expression of renal MR and HSD11β2 (11β-hydroxysteroid dehydrogenase type 2) that determines the availability of MR to aldosterone is significantly higher in AngII-infused female rats than in males. ENaC activity is ≈2× lower in spironolactone-treated AngII-infused female rats than in males. Reduced ENaC activity in AngII-infused female rats on spironolactone correlates with increased interaction with ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2), targeting ENaC for degradation. MR-ENaC axis is the primary determinant of excessive renal sodium reabsorption and an attractive antihypertensive target in female rats with AngII-hypertension, but not in males.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass-spectrometry-based profiling, metabolomics, expression assays and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor (ArsR) superfamily of transcriptional regulators where RSS and H2O2 sensors segregate into distinct clusters. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity and or DNA-dissociation following treatment with GSSG or H2O2. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Studying the interaction between the new neodymium (Nd) complex with the ligand of 1,10-phenanthroline with FS-DNA and BSA.

To learn more about the chemotherapeutic and pharmacokinetic properties of a neodymium complex containing 1,10-phenanthroline (dafone), In vitro binding was investigated with bovine serum albumin and fish-salmon DNA, using a variety of molecular modeling research and biophysical approaches. A variety of spectroscopic techniques including fluorescence and absorption were used to investigate the interplay between DNA/BSA and the neodymium complex. The findings revealed that the Nd complex had a high affinity for BSA and DNA interplays through van der Waals powers. In addition, the binding of the Nd complex to FS-DNA mainly in the groove binding mode clearly reflects with iodide quenching studies, ethidium bromide (EtBr) exclusion assay, ionic strength effect, and viscosity studies. It was observed that the Nd complex binds to FS-DNA through a minor groove with 3.81 × 105(M-1). Also, Kb for BSA at 298K was 5.19×105(M-1), indicating a relatively high affinity of the Nd complex for DNA and BSA. In addition, a competitive study of a docking investigation revealed that the neodymium complex interacts at BSA site III. The results obtained from the binding calculations are well consistent with the experimental findings. Also, cytotoxicity studies of Nd complex were performed in MCF-7 and A-549 cell lines and the results show that this new complex has a selective inhibitory effect on the growth of various cancer cells.

Adhesion tunes speed and persistence by coordinating protrusions and extracellular matrix remodeling.

Cell migration through 3D environments is essential to development, disease, and regeneration processes. Conceptual models of migration have been developed primarily on the basis of 2D cell behaviors, but a general understanding of 3D cell migration is still lacking due to the added complexity of the extracellular matrix. Here, using a multiplexed biophysical imaging approach for single-cell analysis of human cell lines, we show how the subprocesses of adhesion, contractility, actin cytoskeletal dynamics, and matrix remodeling integrate to produce heterogeneous migration behaviors. This single-cell analysis identifies three modes of cell speed and persistence coupling, driven by distinct modes of coordination between matrix remodeling and protrusive activity. The framework that emerges establishes a predictive model linking cell trajectories to distinct subprocess coordination states.

Anticancer Behavior of Pyrrolidine-Based Palladium(II) Complexes and Biophysical Approach on Their DNA, BSA Binding Activity, Molecular Docking, and DFT Study.

A series of pyrrolidine-based Pd(II) complexes, [Pd(AEP)Cl2] (C-1), [Pd(AEP)(OH2)2]2+(C-2), [Pd(AEP)(L-cys)]+ (C-3), [Pd(AEP)(N-ac-L-cys)] (C-4), [Pd(AEP)(GSH)] (C-5), and [Pd(AEP)(DL-meth)]2+ (C-6) (where, AEP = 1-(2-aminoethyl)pyrrolidine, L-cys = l-cysteine, N-ac-L-cys = N-acetyl-l-cysteine, GSH = glutathione, and DL-meth = dl-methionine), as anticancer drug candidates have been synthesized and characterized. The DNA binding property of the complexes was executed by gel electrophoresis and spectrophotometric and viscometric methods, and their interaction with BSA was also investigated by various spectroscopic methodologies. The binding activity of the Pd(II) complexes with DNA and BSA were assessed to evaluate their binding mode and binding constants. Molecular docking was performed to correlate with the experimental results on the interaction of the complexes with DNA and BSA. The changes in the microenvironmental and structural properties of BSA are monitored by a synchronous and 3D fluorescence study. The structural properties were evaluated by DFT and TD-DFT studies. The anticarcinogenic activity of the Pd(II) complexes was assessed by PASS prediction software to corroborate with the experimental results of the anticancer activity of the complexes. The ROS generation in cancer cell lines has been investigated, and the cell death mechanism through apoptosis was confirmed by measuring the protein expression. All these complexes have excellent anticancer activity compared to ancillary ligands. The cancer cell line (HCT116) shows almost similar or better cell inhibition activity when treated with the Pd(II) complexes compared to cisplatin, whereas the adverse effect is minimum on a normal cell (NKE). Both the Pd(II) and Pt(II) complexes carrying the same ligands reveal almost similar antiproliferative activity.

Binding characteristics and conformational changes in alpha-2-macroglobulin by the dietary flavanone naringenin: biophysical and computational approach

In the present study, we investigated the interaction of alpha-2-macroglobulin (α2M) with naringenin using multi-spectroscopic, molecular docking, and molecular simulation approaches to identify the functional changes and structural variations in the α2M structure. Our study suggests that naringenin compromised α2M anti-proteinase activity. The results of absorption spectroscopy and fluorescence measurement showed that naringenin-α2M formed a complex with a binding constant of (kb)∼104, indicative of moderate binding. The value of ΔG° in the binding indicates the process to be spontaneous and the major force responsible to be hydrophobic interaction. The findings of FRET reveal the binding distance between naringenin and the amino acids of α2M was 2.82 nm. The secondary structural analysis of α2M with naringenin using multi-spectroscopic methods like synchronous fluorescence, red-edge excitation shift (REES), FTIR, and CD spectra further confirmed the significant conformational alterations in the protein. Molecular docking approach reveals the interactions between naringenin and α2M to be hydrogen bonds, van der Waals forces, and pi interactions, which considerably favour and stabilise the binding. Molecular dynamics modelling simulations also supported the steady binding with the least RMSD deviations. Our study suggests that naringenin interacts with α2M to alter its confirmation and compromise its activity. Communicated by Ramaswamy H. Sarma

The 5C's of soil security guiding realization of ecosystem services in line with the UN-SDGs

The UN Sustainable Development Goals (SDGs) have a deadline of 2030 so, restricting attention to agriculture, development and implementation of management measures, aimed at reaching the goals, is urgent. Even though the current 159 very broad targets and 234 indicators for the SDGs don't mention soils, soils play a key role when addressing indicators 2.4.1 and 15.3.1 aimed at “sustainable agriculture” and “degraded land”, by introducing operational methods to assess soil health and interdisciplinary ecosystem services in line with the SDGs. Application of the soil security concept, combining biophysical and socioeconomic approaches, is essential for sustainability research where dominant socioeconomic considerations by stakeholders should frame the biophysical analysis. If stakeholders don't embrace SDG-inspired innovative management, nothing will materialize in the real world. Much research is still needed to address inter- and transdisciplinary sustainability issues but a plea is made at this critical point in time to also apply a relatively straightforward and pragmatic research approach, essentially based on available data and methods, to assess ecosystem services, defining indicators and thresholds, in line with five key SDGs. Reference is made to an exploratory Dutch case study in a:”Living Lab”. The National Soil Health Institute in the US has already pioneered such a pragmatic approach by first emphasizing socioeconomic aspects and by incorporating the soil health analysis in regenerative farm management that is recognized by farmers. But the five ecosystem services will still have to be assessed here to maintain the link with the SDGs and the policy arena. The 5C's of soil security apply very well at farm level. The soil science profession will benefit from showing specific examples of “Living Labs” where five thresholds of ecosystem services are met, creating: ”Lighthouses” showing examples of achieved sustainable development in which soil health plays a key role as demonstrated by specific measurements.

Elucidating Residue-Level Determinants Affecting Dimerization of Ebola Virus Matrix Protein Using High-Throughput Site Saturation Mutagenesis and Biophysical Approaches.

The Ebola virus (EBOV) is a filamentous virus that acquires its lipid envelope from the plasma membrane of the host cell it infects. EBOV assembly and budding from the host cell plasma membrane are mediated by a peripheral protein, known as the matrix protein VP40. VP40 is a 326 amino acid protein with two domains that are loosely linked. The VP40 N-terminal domain (NTD) contains a hydrophobic α-helix, which mediates VP40 dimerization. The VP40 C-terminal domain has a cationic patch, which mediates interactions with anionic lipids and a hydrophobic region that mediates VP40 dimer-dimer interactions. The VP40 dimer is necessary for trafficking to the plasma membrane inner leaflet and interactions with anionic lipids to mediate the VP40 assembly and oligomerization. Despite significant structural information available on the VP40 dimer structure, little is known on how the VP40 dimer is stabilized and how residues outside the NTD hydrophobic portion of the α-helical dimer interface contribute to dimer stability. To better understand how VP40 dimer stability is maintained, we performed computational studies using per-residue energy decomposition and site saturation mutagenesis. These studies revealed a number of novel keystone residues for VP40 dimer stability just adjacent to the α-helical dimer interface as well as distant residues in the VP40 CTD that can stabilize the VP40 dimer form. Experimental studies with representative VP40 mutants in vitro and in cells were performed to test computational predictions that reveal residues that alter VP40 dimer stability. Taken together, these studies provide important biophysical insights into VP40 dimerization and may be useful in strategies to weaken or alter the VP40 dimer structure as a means of inhibiting the EBOV assembly.

Petrale sole transboundary connectivity and settlement success: a biophysical approach

Connectivity between inferred spawning areas and potential settlement areas of the petrale sole (Eopsetta jordani) was explored in the California Current System (CCS) using an individual-based model (IBM) coupled with the ROMS hydrodynamic model for the period 1988-2008. The IBM modeled pelagic early life stages, including egg and larval development, growth, natural mortality and settlement into benthos. Eggs were released within discrete spawning grounds identified from the winter fishery logbook data. Potential settlement areas were defined based on bathymetrical criteria and juvenile (2-year old) distribution from a groundfish bottom trawl survey. The influence of cross-shelf and alongshore advection on the transport and connectivity between spawning and benthic settlement areas was explored by identifying the location of juveniles (22 mm length) among the potential settlement areas. The most important spawning regions varied over time with between 4 and 19% of spawned individuals successfully settling, mostly ranging from off northern Washington to northern Oregon. The strong influence of northward alongshore transport resulted in transboundary transport of pelagic life stages from U.S. spawning grounds to inner shelf settlement areas in Canadian waters, with 33.9-70.4% (average 49.7 ± 9.6) of annual successful juveniles settling in Canada. Interannual variability in juvenile settlement success suggests that mesoscale (100-200 km) oceanographic structures play a major role defining pelagic juvenile transport trajectories. While, in some years, the presence of coastal cyclonic eddies can retain juveniles off Oregon, the strong northward transport supplies a large number of juveniles to Moresby and Vancouver island, clearly showing that petrale sole off the west coast of North America are a transboundary stock with important settlement areas off the coast of British Columbia, thus extension of this study into Canadian waters is particularly relevant.

Identification of residues critical for the extension of Munc18-1 domain 3a

BackgroundNeurotransmitter release depends on the fusion of synaptic vesicles with the presynaptic membrane and is mainly mediated by SNARE complex assembly. During the transition of Munc18-1/Syntaxin-1 to the SNARE complex, the opening of the Syntaxin-1 linker region catalyzed by Munc13-1 leads to the extension of the domain 3a hinge loop, which enables domain 3a to bind SNARE motifs in Synaptobrevin-2 and Syntaxin-1 and template the SNARE complex assembly. However, the exact mechanism of domain 3a extension remains elusive.ResultsHere, we characterized residues on the domain 3a hinge loop that are crucial for the extension of domain 3a by using biophysical and biochemical approaches and electrophysiological recordings. We showed that the mutation of residues T323/M324/R325 disrupted Munc13-1-mediated SNARE complex assembly and membrane fusion starting from Munc18-1/Syntaxin-1 in vitro and caused severe defects in the synaptic exocytosis of mouse cortex neurons in vivo. Moreover, the mutation had no effect on the binding of Synaptobrevin-2 to isolated Munc18-1 or the conformational change of the Syntaxin-1 linker region catalyzed by the Munc13-1 MUN domain. However, the extension of the domain 3a hinge loop in Munc18-1/Syntaxin-1 was completely disrupted by the mutation, leading to the failure of Synaptobrevin-2 binding to Munc18-1/Syntaxin-1.ConclusionsTogether with previous results, our data further support the model that the template function of Munc18-1 in SNARE complex assembly requires the extension of domain 3a, and particular residues in the domain 3a hinge loop are crucial for the autoinhibitory release of domain 3a after the MUN domain opens the Syntaxin-1 linker region.

High light-induced changes in whole-cell proteomic profile and its correlation with the organization of thylakoid super-complex in cyclic electron transport mutants of Chlamydomonas reinhardtii.

Light and nutrients are essential components of photosynthesis. Activating the signaling cascades is critical in starting adaptive processes in response to high light. In this study, we have used wild-type (WT), cyclic electron transport (CET) mutants like Proton Gradient Regulation (PGR) (PGRL1), and PGR5 to elucidate the actual role in regulation and assembly of photosynthetic pigment-protein complexes under high light. Here, we have correlated the biophysical, biochemical, and proteomic approaches to understand the targeted proteins and the organization of thylakoid pigment-protein complexes in the photoacclimation. The proteomic analysis showed that 320 proteins were significantly affected under high light compared to the control and are mainly involved in the photosynthetic electron transport chain, protein synthesis, metabolic process, glycolysis, and proteins involved in cytoskeleton assembly. Additionally, we observed that the cytochrome (Cyt) b6 expression is increased in the pgr5 mutant to regulate proton motive force and ATPase across the thylakoid membrane. The increased Cyt b6 function in pgr5 could be due to the compromised function of chloroplast (cp) ATP synthase subunits for energy generation and photoprotection under high light. Moreover, our proteome data show that the photosystem subunit II (PSBS) protein isoforms (PSBS1 and PSBS2) expressed more than the Light-Harvesting Complex Stress-Related (LHCSR) protein in pgr5 compared to WT and pgrl1 under high light. The immunoblot data shows the photosystem II proteins D1 and D2 accumulated more in pgrl1 and pgr5 than WT under high light. In high light, CP43 and CP47 showed a reduced amount in pgr5 under high light due to changes in chlorophyll and carotenoid content around the PSII protein, which coordinates as a cofactor for efficient energy transfer from the light-harvesting antenna to the photosystem core. BN-PAGE and circular dichroism studies indicate changes in macromolecular assembly and thylakoid super-complexes destacking in pgrl1 and pgr5 due to changes in the pigment-protein complexes under high light. Based on this study, we emphasize that this is an excellent aid in understanding the role of CET mutants in thylakoid protein abundances and super-complex organization under high light.