Monomeric Conformation Research Articles

Spectroscopic, structural and conformational investigations of the major constituent of the clove oil: Eugenol

Molecular structures of the 12 possible conformers of eugenol were optimized at the B3LYP/6–311++G** level available with the Gaussian 09 software. IR and Raman spectra of eugenol were measured and analysed in light of the computed quantities. The interpretation of the IR spectrum in the OH stretching region suggested the existence of eugenol in the dimeric form. Five dimeric structures of the lowest energy monomeric conformer (C-I) were found to have energy difference below 300K. The complete observed IR and Raman spectra could be explained in terms of the computed spectra of the five lowest energy dimers of C-I. UV–vis absorption spectrum was measured in the range 200–600 nm and analysed with the help of computed parameters. Moreover, MEP plots and HOMO-LUMO analysis were carried out. Different computed quantities/ parameters for C-I and its 5 dimers are compared. In addition, barrier heights for different tops, thermodynamic functions and bioactive scores of the eugenol molecule were determined.

E46K Mutation of α-Synuclein Preorganizes the Intramolecular Interactions Crucial for Aggregation.

Aggregation of α-synuclein is central to the pathogenesis of Parkinson's disease. The most toxic familial mutation E46K accelerates the aggregation process by an unknown mechanism. Herein, we provide a clue by investigating the influence of E46K on monomeric α-synuclein and its relation to aggregation with molecular dynamics simulations. The E46K mutation suppresses β-sheet structures in the N-terminus while promoting those at the key fibrillization region named NACore. Even though WT and E46K monomers share conserved intramolecular interactions with fibrils, E46K abolishes intramolecular contacts within the N-terminus which are present in the WT monomer but absent in fibrils. Network analysis identifies residues 36-53 as the interaction core of the WT monomer. Upon mutation, residues 36-46 are expelled to water due to aggravated electrostatic repulsion in the 43KTKK46 segment. Instead, NACore (residues 68-78) becomes the interaction hub and connects preceding residues 47-56 and the C-terminus. Consequently, residues 47-95 which belong to the fibril core form more compact β-sheets. Overall, the interaction network of E46K is more like fibrils than WT, stabilizing the fibril-like conformations. Our work provides mechanistic insights into the faster aggregation of the E46K mutant. It implies a close link between monomeric conformations and fibrils, which would spur the development of therapeutic strategies.

Conformational fluctuations and phases in fused in sarcoma (FUS) low-complexity domain.

The well-known phenomenon of phase separation in synthetic polymers and proteins has become a major topic in biophysics because it has been invoked as a mechanism of compartment formation in cells, without the need for membranes. Most of the coacervates (or condensates) are composed of Intrinsically Disordered Proteins (IDPs) or regions that are structureless, often in interaction with RNA and DNA. One of the more intriguing IDPs is the 526-residue RNA-binding protein, Fused in Sarcoma (FUS), whose monomer conformations and condensates exhibit unusual behavior that are sensitive to solution conditions. By focussing principally on the N-terminus low-complexity domain (FUS-LC comprising residues 1-214) and other truncations, we rationalize the findings of solid-state NMR experiments, which show that FUS-LC adopts a non-polymorphic fibril structure(core-1) involving residues 39-95, flanked by fuzzy coats on both the N- and C-terminal ends. An alternate structure (core-2), whose free energy is comparable to core-1, emerges only in the truncated construct (residues 110-214). Both core-1 and core-2 fibrils are stabilized by a Tyrosine ladder as well as hydrophilic interactions. The morphologies (gels, fibrils, and glass-like) adopted by FUS seem to vary greatly, depending on the experimental conditions. The effect of phosphorylation is site-specific. Simulations show that phosphorylation of residues within the fibril has a greater destabilization effect than residues that are outside the fibril region, which accords well with experiments. Many of the peculiarities associated with FUS may also be shared by other IDPs, such as TDP43 and hnRNPA2. We outline a number of problems for which there is no clear molecular explanation.

Endocytic pathways of pathogenic protein aggregates in neurodegenerative diseases.

Endocytosis is the fundamental uptake process through which cells internalize extracellular materials and species. Neurodegenerative diseases (NDs) are characterized by a progressive accumulation of intrinsically disordered protein species, leading to neuronal death. Misfolding in many proteins leads to various NDs such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and other disorders. Despite the significance of disordered protein species in neurodegeneration, their spread between cells and the cellular uptake of extracellular species is not entirely understood. This review discusses the major internalization mechanisms of the different conformer species of these proteins and their endocytic mechanisms. We briefly introduce the broad types of endocytic mechanisms found in cells and then summarize what is known about the endocytosis of monomeric, oligomeric and aggregated conformations of tau, Aβ, α-Syn, Huntingtin, Prions, SOD1, TDP-43 and other proteins associated with neurodegeneration. We also highlight the key players involved in internalizing these disordered proteins and the several techniques and approaches to identify their endocytic mechanisms. Finally, we discuss the obstacles involved in studying the endocytosis of these protein species and the need to develop better techniques to elucidate the uptake mechanisms of a particular disordered protein species.

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Crystal structures and solution conformations of HtrA from Helicobacter pylori reveal pH-dependent oligomeric conversion and conformational rearrangements

Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects over 50 % of the world's population, making it a major risk factor for chronic gastritis, ulcer diseases of the stomach and duodenum, MALT lymphoma, and gastric cancer. The clinical consequences of H. pylori infection are closely linked with the expression of virulence factors secreted by the bacterium. One such virulence factor is high temperature requirement A (HtrA), which possesses chaperone and serine protease activity. In the host stomach, HtrA secreted from H. pylori (HpHtrA) disrupts intercellular adhesions by cleaving epithelial adhesion proteins including E-cadherin and desmoglein-2. This disruption causes intercellular junctions to open, allowing the bacterium to pass through the epithelial barrier, access the intercellular space, and colonize the gastric mucosa. HtrA proteases are well known for their structural complexity, reflected in their diverse oligomer forms and multi-tasking activities in both prokaryotes and eukaryotes. In this study, we determined crystal structures and solution conformations of HpHtrA monomer and trimer, which revealed large domain rearrangements between them. Notably, this is the first report of a monomeric structure in the HtrA family. We further found a pH-dependent dynamic trimer-to-monomer conversion and concurrent conformational changes that seem closely linked with a pH-sensing ability through the protonation of certain Asp residues. These results advance our understanding of the functional roles and the related mechanisms of this protease in bacterial infection, which may shed light on the development of HtrA-targeted therapies for H. pylori-associated diseases.

Tailoring amide N-substitution to direct liquid crystallinity in benzanilide-based dimers

The design of liquid crystalline dimers containing tertiary benzanilide groups has proved challenging due to their strong preference for the E amide conformation. In this work we have investigated the effect of different N-substituents on the amide conformation of model benzanilide monomers and found this had effectively no effect on conformation. Based on this observation, we investigated the effect of introducing large, flexible decyl chains at the amide nitrogen and compare their properties to the corresponding benzanilide-based dimers with lateral methyl substituents. The introduction of the decyl chain decreases the melting temperatures, and the tendency to crystallise, promoting glassy behaviour. This allows for nematic behaviour to be seen to much lower temperatures.

Heterologous expression, purification, and characterization of a recombinant Cordyceps militaris lipase from Candida rugosa-like family in Pichia pastoris

Multiple sequence alignments of three lipase isoforms from the filamentous fungus, Cordyceps militaris, have revealed that the deduced protein from their common sequence belongs to the Candida rugosa lipase-like group. To express the protein in its active form, recombinant lipase from C. militaris (rCML) was extra cellularly expressed in Pichia pastoris X-33 after removing its signal peptide. Purified rCML was a stable monomeric protein with a molecular mass of 90 kDa, and was highly N-mannosylated compared to the native protein (69 kDa). The catalytic efficiency (kcat/Km) of rCML was greater than the native protein (1244.35 ± 50.88 and 1067.17 ± 29.07 mM−1·min−1, respectively), yet they had similar optimal pH values and temperatures (40 °C and pH 7.0–7.5), and showed preferences for Tween esters and short-chain triacylglycerols. Despite its monomeric conformation, interfacial activation was not observed for rCML, unlike the classical lipases. From the structural model of rCML, the binding pocket of rCML was predicted as a funnel-like structure consisting of a hollow space and an intramolecular tunnel, which is typical of C. rugosa lipase-like lipases. However, a blockage shortened the tunnel to 12–15 Å, which endows strict short-chain selectivity towards triacylglycerols and a perfect match for tricaproin (C6:0). The limited depth of the tunnel may enable accommodation of triacylglycerols with medium-to-long-chain fatty acids, which differentiates rCML from other C. rugosa lipase-like lipases with broad substrate specificities.

Identification and functional characterisation of N-acetylglucosamine kinase from Helicoverpa armigera divulge its potential role in growth and development via UDP-GlcNAc salvage pathway

N-acetylglucosamine kinase (NAGK), a major enzyme of sugar-kinase/Hsp70/actin superfamily, catalyses the conversion of N-acetylglucosamine to N-acetylglucosamine-6-phosphate, the first step leading to the salvage synthesis of uridine diphosphate N-acetylglucosamine. Here, we present the first report on identification, cloning, recombinant expression and functional characterisation of NAGK from Helicoverpa armigera (HaNAGK). The purified soluble HaNAGK exhibited a molecular mass of ∼39 kDa with monomeric conformation. It catalysed the sequential transformation of GlcNAc into UDP-GlcNAc, indicating its role as the initiator of UDP-GlcNAc salvage pathway. HaNAGK exhibited ubiquitous expressions across all the developmental stages and major tissues of H. armigera. The gene was significantly upregulated (80 %; p < 0.01) by the moulting hormone 20-hydroxyecdysone and significantly downregulated (89 %; p < 0.001) by the chitin synthesis inhibitor novaluron, indicating its involvement in ecdysis and chitin metabolism. Furthermore, RNAi of HaNAGK caused poor weight gain, deformed insect bodies, aberrant metamorphosis and pronounced wing abnormalities in >55 % of surviving adults, while recording 7.79 ± 1.52 % and 24.25 ± 7.21 % mortality during larval and pupal stages, respectively. Altogether, the present findings suggest that HaNAGK plays a crucial role in the growth and development of H. armigera and thus, could be considered as a compelling gene of interest while formulating novel pest management strategies.

Abstract PR02: Factors modulating RAF dimerization downstream of RAS – A mechanistic overview

Abstract The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. The activation of this pathway is tightly regulated with appropriate spatial and temporal signaling cues. The central step in activation of this pathway is the dimerization of RAF kinases. Recent advances in biochemical, enzymatic and structural characterization of various multiprotein complexes in this pathway provide insight into various modes of RAF activity modulation. Kinase RAF and its substrate MEK exist as a pre-formed complex prior to pathway activation. Multiple factors prevent MEK phosphorylation by RAF in this pre-formed complex, including phosphorylation of a Serine residue N-terminal to the kinase domain, which enables 14-3-3 to trap RAF as a monomer. One of our intriguing findings is that cellular ATP exerts a negative regulatory effect on RAF kinase and stabilizes the inactive conformation of RAF monomer. We characterize this negative regulatory effect of ATP of RAF kinase structurally by solving a RAF-ATP complex and enzymology. Upon membrane recruitment of RAF by RAS-GTP, dephosphorylation of the above-mentioned serine residue in RAF occurs by a multiprotein complex composed of phosphatase PP1C, a scaffolding protein SHOC2 and RAS-GTP. We solved the structure of this RAS-SHOC2-PP1C complex and performed a thorough enzymatic analysis of this complex for RAF dephosphorylation. Our results show that RAF specificity is determined by SHOC2 and RAS GTP is responsible for spatial localization both inactive RAF and SHOC2/PP1C to the membrane. This dephosphorylation results in RAF being stabilized as a dimer by 14-3-3, which now binds to phospho-serine residues C-terminal to the kinase domain (S729 in BRAF) in 2 RAF molecules. We solve a structure of the RAF-14-3-3 complex. 14-3-3 induced RAF dimerization increases activity of RAF ~500 fold. Further, oncogenic mutations in RAF and MEK occur basally in tumors, and additional mutations are induced upon pathway inhibitor treatment in the clinic, especially for recent KRAS-G12C treatments. These mutations include RAF mutations that either activate or inactivate the kinase activity of the mutated RAF, but nevertheless result in pathway activation. Our data suggests that these mutations appear to tilt the RAF monomer-dimer equilibrium towards RAF dimer. The molecular mechanism of how the MEK mutations active the pathway in poorly understood. Biochemical and structural characterization of these mutations occurring in MEK kinases suggests that the mechanism of activation is also likely functioning via promoting RAF dimerization. This RAF dimer promotion appears to mechanistically work by relieving the negative regulatory effect of RAF by ATP to result in the “just-right” amount of pathway activation to sustain tumor growth. A comprehensive understanding of the various factors modulating RAF dimerization in the context of normal cell and cancer cells that depend on this pathway can pave the way for more precise interventions for treatment of cancers that depend on the RAS pathway. Citation Format: Jawahar Sudhamsu, Nicholas Liau, Timothy Wendorff, Saeed Izadi, Luca Gerosa. Factors modulating RAF dimerization downstream of RAS – A mechanistic overview [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR02.

The Use of Novel Antibodies to Characterize B cells in Health and Disease

IntroductionCD38, a glycoprotein ectoenzyme found on the surface of majority of hematopoietic cells, can exist in monomeric or multimeric conformations and enzymatic activity correlates with multimerization. During human B cell differentiation, CD38 expression levels are low on naïve and memory B cells, intermediate on transitional cells, and high on antibody-secreting plasmablasts and plasma cells. Conventional anti-CD38 (C-CD38) antibodies, recognize all CD38 formations and are not selective for identification of plasmablasts, thus requiring the use of additional antibodies including CD27. In contrast, a novel sea lamprey derived monoclonal VLRB antibody, tetVLRB MM3 (V-CD38), recognizes an epitope likely located within the catalytic site of active multimeric CD38 and therefore might be more specific to plasmablasts, which express active CD38. ObjectivesCharacterize the B cells recognized by V-CD38 in the peripheral blood of individuals with or without inborn errors of immunity (IEI). MethodsB cell subsets were characterized as described in figure 1 using conventional antibodies to CD3, CD19, IgM, CD27, C-CD38, and by V-CD38 generated by a collaborator. Results were presented as means with standard deviations (SD). Correlations were calculated using the Pearson’s Correlation Coefficient formula. ResultsTo validate our identification of plasmablasts with V-CD38, samples from patients with X-linked agammaglobulinemia lacking B cells, and tonsils rich in plasmablasts were used as negative and positive controls, respectively. Analysis of samples from 16 individuals revealed nearly identical (r = 0.99, p <.00001) proportions of CD27hi plasmablasts recognized by V-CD38 (0.51%+/-0.74%) and C-CD38 (0.49%+/-0.70%), suggesting that all cells expressed multimeric CD38. In contrast, only 45.28% ±16.19% of transitional IgMhighCD27-C-CD38int B cells were also recognized by V-CD38, (2.52%+/–3.62% and 1.15%+/–1.75%, respectively), a finding that was consistent across samples (r = 0.98, p <.00001). Preliminary studies in three IEI patients demonstrated markedly reduced plasmablasts (0.035%+/–0.026%), class-switched memory B cells (1.25% +/–1.52%) and transitional cells (1.25%+/–1.06%) compared to healthy controls (0.59%+/–0.74%, 15.29%+/–8.95%, and 2.81%+/–3.96% respectively). [Display omitted] ConclusionsWhile C-CD38 and V-CD38 antibodies can identify plasmablasts in peripheral blood, V-CD38 allows more selective detection of CD38 on plasmablasts. Some circulating transitional cells also express multimeric CD38. Additional studies will determine whether V-CD38 can better identify patients suffering from abnormal plasmablast formation and impaired production of antibodies.

Abstract A036: Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation

Abstract The RAS-RAF-MEK-ERK signaling axis is upregulated in many human cancers due to its central role in cell growth, differentiation and survival. Oncogenic transformation frequently arises from RAS, BRAF and MEK mutations that activate the pathway. In the clinic, similar transformations also recur as resistance mutations to KRAS.G12C inhibitor treatment. BRAF mutations at V600 (Class I) activate BRAF by releasing the kinase’s activation segment and rendering BRAF activity dimerization-independent. Yet, it remains unclear how a diverse set of dimerization-dependent BRAF mutations (Class II/III) activate the pathway. MEK mutations are assumed to bypass the RAF node entirely. We previously reported a 2.9-Å-resolution crystal structure of human BRAF kinase domain (BRAFKD) in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that stabilize an inactive, pre-signaling monomeric conformation of BRAFKD and explain how ATP breaks RAF dimers in solution. Surprisingly, the β3-αC loop of MEK provides additional stabilizing interactions to the BRAF-bound AMP-PCP molecule. Utilizing an in vitro RAF dimerization assay, we find that MEK not only modulates RAF-RAF dimerization, it also enhances both the dimer-breaking effect ATP and the dimer-forming effect of paradoxical activator molecules, such as GDC-0879. Structural analysis reveals that all common oncogenic BRAF mutations alter key interactions with ATP that stabilize the inactive monomer conformation. We find ATP is unable to break RAF dimers containing Class I, II or III mutations, confirming these mutants counteract the inhibitory effects of ATP binding by lowering the threshold for RAF dimerization and thus pathway activation. We further find that oncogenic MEK mutants, in particular β3-αC loop deletions, favor RAF-RAF dimerization, even in the presence of ATP. Our study establishes a framework for rationalizing oncogenic BRAF mutations, and suggests that oncogenic MEK mutants can act, at least in part, by promoting RAF dimerization. This model provides a better understanding of activation mechanisms in the context of constantly evolving resistance mutations where oncogenesis continues to depend on the MAPK pathway. Citation Format: Timothy J. Wendorff, Jennifer Kung, Jawahar Sudhamsu. Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A036.

Escherichia coli FtsZ molecular dynamics simulations

Earlier molecular dynamics studies of the FtsZ protein revealed that the protein has high intrinsic flexibility which the crystal structures cannot reveal. However, the input structure in these simulation studies was based on the available crystal structure data and therefore, the effect of the C-terminal Intrinsically Disordered Region (IDR) of FtsZ could not be observed in any of these studies. Recent investigations have revealed that the C-terminal IDR is crucial for FtsZ assembly in vitro and Z ring formation in vivo. Therefore, in this study, we simulated FtsZ with the IDR. Simulations of the FtsZ monomer in different nucleotide bound forms (without nucleotide, GTP, GDP) were performed. In the conformations of FtsZ monomer with GTP, GTP binds variably with the protein. Such a variable interaction with the monomer has not been observed in any previous simulation studies of FtsZ and not observed in crystal structures. We found that central helix bends towards the C-terminal domain in the GTP bound form, hence, making way for polymerization. A nucleotide dependent shift/rotation of the C-terminal domain was observed in simulation time averaged structures. Communicated by Ramaswamy H. Sarma

2D-monolayer formation of 3-monoacyl-sn-glycerols at the air/water interface. A comprehensive quantum chemical approach

Thermodynamic and structural aspects of the monolayer film formation of 3-monoacyl-sn-glycerol homologous series with the alkyl chain length n = 7–18 at the air/water interface are considered. The calculations are carried out within the framework of the semiempirical quantum chemical method PM3 and supported experimentally using surface pressure-area per molecule (π–A) isotherms, Brewster angle microscopy (BAM), as well as grazing incidence X-ray diffraction analysis (GIXD). A conformational analysis of 3-monoacyl-sn-glycerol monomers reveals three stable conformations of monomers with Monomer 1 as the possible basis for the construction of the most energetically advantageous structures of small clusters, which subsequently can form a 2D monolayer. Optimized tetramer structure of 3-monoacyl-sn-glycerol is considered as a unit cell of the 2D film with geometric parameters: a= 4.82 Å, b= 4.92 Å, and the angle between them θ = 91°. The molecular tilt angle of surfactant molecules with respect to the normal to the interface is 23.5°. These structural parameters are in good agreement with existing experimental data. The spontaneous clusterization threshold of 3-monoacyl-sn-glycerol at the air/water interface is 16 carbon atoms in the alkyl chain at standard temperature. The temperature dependences of the Ac area per film molecule at the beginning of the liquid-expanded–liquid-condensed (LE–LC) phase transition are analyzed. The values of the Ac change per 1 °C for 3-monoacyl-sn-glycerol are commensurable with corresponding values of other surfactant classes confirming the dominating role of intermolecular CH···HC interactions in the clusterization energy of surfactants.

Antibodies for β2-Microglobulin and the Heavy Chains of HLA-E, HLA-F, and HLA-G Reflect the HLA-Variants on Activated Immune Cells and Phases of Disease Progression in Rheumatoid Arthritis Patients under Treatment

Rheumatoid arthritis (RA) is a progressive, inflammatory, autoimmune, symmetrical polyarticular arthritis. It is characterized by synovial infiltration and activation of several types of immune cells, culminating in their apoptosis and antibody generation against "altered" autoantigens. β2-microglobulin (β2m)-associated heavy chains (HCs) of HLA antigens, also known as closed conformers (Face-1), undergo "alteration" during activation of immune cells, resulting in β2m-free structural variants, including monomeric open conformers (Face-2) that are capable of dimerizing as either homodimers (Face-3) or as heterodimers (Face-4). β2m-free HCs uncover the cryptic epitopes that can elicit antibodies (Abs). We report here the levels of IgM and IgG Abs against both β2m and HCs of HLA-E, HLA-F, and HLA-G in 74 RA patients receiving immunosuppressive drugs. Anti-β2m IgM was present in 20 of 74 patients, whereas anti-β2m IgG was found in only 8 patients. Abs against β2m would be expected if Abs were generated against β2m-associated HLA HCs. The majority of patients were devoid of either anti-β2m IgM or IgG but had Abs against HCs of different HLA-Ib molecules. The paucity of anti-β2m Abs in this cohort of patients suggests that Abs were developed against β2m-free HLA HCs, such as Face-2, Face-3, and Face-4. While 63 of 68 patients had IgG Abs against anti-HLA-F HCs, 36 and 50 patients showed IgG Ab reactivity against HLA-E and anti-HLA-G HCs, respectively. Evidently, anti-HLA-F HC Abs are the most predominant anti-HLA-Ib HC IgG Abs in RA patients. The incidence and intensity of Abs against HLA-E, HLA-F, and HLA-G in the normal control group were much higher than those observed in RA patients. Evidently, the lower level of Abs in RA patients points to the impact of the immunosuppressive drugs on these patients. These results underscore the need for further studies to unravel the nature of HLA-F variants on activated immune cells and synoviocytes of RA patients.

Conformation-dependent dynamic organic phosphorescence through thermal energy driven molecular rotations

Organic room-temperature phosphorescent (RTP) materials exhibiting reversible changes in optical properties upon exposure to external stimuli have shown great potential in diverse optoelectronic fields. Particularly, dynamic manipulation of response behaviors for such materials is of fundamental significance, but it remains a formidable challenge. Herein, a series of RTP polymers were prepared by incorporating phosphorescent rotors into polymer backbone, and these materials show color-tunable persistent luminescence upon excitation at different wavelengths. Experimental results and theoretical calculations revealed that the various molecular conformations of monomers are responsible for the excitation wavelength-dependent (Ex-De) RTP behavior. Impressively, after gaining insights into the underlying mechanism, dynamic control of Ex-De RTP behavior was achieved through thermal energy driven molecular rotations of monomers. Eventually, we demonstrate the practical applications of these amorphous polymers in anti-counterfeiting areas. These findings open new opportunities for the control of response behaviors of smart-responsive RTP materials through external stimuli rather than conventional covalent modification method.

Early-Stage Oligomerization of Prion-like Polypeptides Reveals the Molecular Mechanism of Amyloid-Disrupting Capacity by Proline Residues.

Proline cis/trans isomerization governs protein local conformational changes via its local mechanical rigidity. The amyloid-disrupting capacity of proline is widely acknowledged; however, the molecular mechanism is still not clear. To understand how proline residues in polypeptide chains influence amyloid propensity, we study several truncated sequences of the TDP-43 C-terminal region (287-322) and their triple proline variants (308PPP310). We use coarse-grained molecular simulation to study the time evolution of the process of aggregation in the early stages in an effective high-concentration condition (∼25 mM). This ensures the long time scales for protein association at laboratory concentrations. We use several experimentally determined structure templates as initial structures of monomer conformations. We carry out oligomer size analysis and cluster analysis, along with several structural measures, to characterize the size distributions of oligomers and their morphological/structural properties. We show that average oligomer size is not a good indicator of amyloid propensity. Structural order and/or morphological properties are better alternatives. We show that proline variants can efficiently maintain the formation of large "ordered" oligomers of shorter truncated sequences, i.e., 307-322. This "order" maintenance is weakened when using longer truncated sequences (i.e., 287-322), leading to the formation of "disordered" oligomers. From an energy trade-off perspective, if the entropic effect is weak (short sequence length), the shape-complementarity of proline variants effectively guides the oligomerization process to form "ordered" oligomer intermediates. This leads to a distinct aggregation pathway that promotes amyloid formation (on-pathway). Strong entropic effects (long sequence length), however, would cause the formation of "disordered" oligomers. This in turn will suppress amyloid formation (off-pathway). The proline shape-complementary effects provide a guided morphological restraint to facilitate the pathways of amyloid formation. Our study supports the importance of structure-based kinetic heterogeneity of prion-like sequence fragments in driving different aggregation pathways. This work sheds light on the role of morphological and structural order of early-stage oligomeric species in regulating amyloid-disrupting capacity by prolines.

Monomeric and dimeric states of human ZO1-PDZ2 are functional partners of the SARS-CoV-2 E protein

The Envelope (E) protein of SARS-CoV-2 plays a key role in virus maturation, assembly, and virulence mechanisms. The E protein is characterized by the presence of a PDZ-binding motif (PBM) at its C-terminus that allows it to interact with several PDZ-containing proteins in the intracellular environment. One of the main binding partners of the SARS-CoV-2 E protein is the PDZ2 domain of ZO1, a protein with a crucial role in the formation of epithelial and endothelial tight junctions (TJs). In this work, through a combination of analytical ultracentrifugation analysis and equilibrium and kinetic folding experiments, we show that ZO1-PDZ2 domain is able to fold in a monomeric state, an alternative form to the dimeric conformation that is reported to be functional in the cell for TJs assembly. Importantly, surface plasmon resonance (SPR) data indicate that the PDZ2 monomer is fully functional and capable of binding the C-terminal portion of the E protein of SARS-CoV-2, with a measured affinity in the micromolar range. Moreover, we present a detailed computational analysis of the complex between the C-terminal portion of E protein with ZO1-PDZ2, both in its monomeric conformation (computed as a high confidence AlphaFold2 model) and dimeric conformation (obtained from the Protein Data Bank), by using both polarizable and nonpolarizable simulations. Together, our results indicate both the monomeric and dimeric states of PDZ2 to be functional partners of the E protein, with similar binding mechanisms, and provide mechanistic and structural information about a fundamental interaction required for the replication of SARS-CoV-2.

Computational Assisted-Molecular Imprinted Polymer Design for Magnetic Solid Phase Extraction of Pinostrobin from Krachai

The Covid-19 pandemic is driving the use of herbal extracts to treat early stages of Covid-19 symptoms, particularly in light of the increased demand for Krachai (Boesenbergia rotunda (L.) Mansf.) with significant pharmacological properties. However, the relevance of establishing extract methods is still necessary for Krachai standardization at an industrial level for continued industrial development of food and health products. The purpose of this work was to develop a unique solid phase extraction with particular recognition produced by imprinting technology on the suface of Fe3O4 nanoparticles that may enable selective absorption of pinostrobin, an active ingredient in Krachai. Pre-polymerization procedure of molecularly imprinted polymer (MIP) nanoparticles specific to pinostrobin was critical to computational aided-design MIP nanoparticles in this preliminary study. The molecular modeling interactions between two mixed functional monomers, methacrylic acid and vinylpyridine, in the presence of pinostrobin, a printed molecule were used by Hyperchem 7.5 software package. The complex was built and simulated in various ratios of template and functional monomer conformations in order to geometrically optimize it to the lowest energy state in the gas phase. The estimated free binding energies of each conformation were calculated using the semi-empirical PM3 simulation method. The results showed that interactions with the ratio of 1:2:2 (pinostrobin: methacrylic acid: vinylpyridine) had the maximized free binding energy when compared to interactions with other ratios. Additionally, the FT-IR approach was

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein.

In Parkinson's disease and other synucleinopathies, α-synuclein misfolds and aggregates. Its intrinsically disordered nature, however, causes it to adopt several meta-stable conformations stabilized by internal hydrogen bonding. Because they interconvert on short timescales, monomeric conformations of disordered proteins are difficult to characterize using common structural techniques. Few techniques can measure the conformations of monomeric α-synuclein, including millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Here, we demonstrate a new approach correlating millisecond HDX-MS data with aggregation kinetics to determine the localized structural dynamics that underpin the self-assembly process in full-length wild-type monomeric α-synuclein. Our custom instrumentation and software enabled measurement of the amide hydrogen-exchange rates on the millisecond timescale for wild-type α-synuclein monomer up to residue resolution and under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal cellular compartments. We applied an empirical correction to normalize measured hydrogen-exchange rates and thus allow comparison between drastically different solution conditions. We characterized the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Applying a correlative approach to connect molecular conformation to aggregation in α-synuclein for the first time, we found that the central C-terminal residues of α-synuclein are driving its nucleation and thus its aggregation. We provide a new approach to link the local structural dynamics of intrinsically disordered proteins to functional attributes, which we evidence with new details on our current understanding of the relationship between the local chemical environment and conformational ensemble bias of monomeric α-synuclein.