scholarly journals Structural Basis of the Function of Yariv Reagent—An Important Tool to Study Arabinogalactan Proteins

2021 ◽  
Vol 8 ◽  
Author(s):  
Tereza Přerovská ◽  
Anna Pavlů ◽  
Dzianis Hancharyk ◽  
Anna Rodionova ◽  
Anna Vavříková ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Plant Defence ◽  
Three Dimensional ◽  
Arabinogalactan Proteins ◽  
Arabinogalactan Protein ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Functional Studies ◽  
Yariv Reagent ◽  
Dynamics Simulations

Arabinogalactan proteins are very abundant, heavily glycosylated plant cell wall proteins. They are intensively studied because of their crucial role in plant development as well as their function in plant defence. Research of these biomacromolecules is complicated by the lack of tools for their analysis and characterisation due to their extreme heterogeneity. One of the few available tools for detection, isolation, characterisation, and functional studies of arabinogalactan proteins is Yariv reagents. Yariv reagent is a synthetic aromatic glycoconjugate originally prepared as an antigen for immunization. Later, it was found that this compound can precipitate arabinogalactan proteins, namely, their ß-D-(1→3)-galactan structures. Even though this compound has been intensively used for decades, the structural basis of arabinogalactan protein precipitation by Yariv is not known. Multiple biophysical studies have been published, but none of them attempted to elucidate the three-dimensional structure of the Yariv-galactan complex. Here we use a series of molecular dynamics simulations of systems containing one or multiple molecules of ß-D-galactosyl Yariv reagent with or without oligo ß-D-(1→3)-galactan to predict the structure of the complex. According to our model of Yariv-galactan complexes, Yariv reagent forms stacked oligomers stabilized by π-π and CH/π interactions. These oligomers may contain irregularities. Galactan structures crosslink these Yariv oligomers. The results were compared with studies in literature.

scholarly journals Online biophysical predictions for SARS-CoV-2 proteins

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Luciano Kagami ◽  
Joel Roca-Martínez ◽  
Jose Gavaldá-García ◽  
Pathmanaban Ramasamy ◽  
K. Anton Feenstra ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Static Structure ◽  
Homologous Proteins ◽  
Structure Based Drug Design ◽  
Protein Protein Interaction ◽  
Link Type ◽  
Dynamics Simulations ◽  
Β Sheet

Abstract Background The SARS-CoV-2 virus, the causative agent of COVID-19, consists of an assembly of proteins that determine its infectious and immunological behavior, as well as its response to therapeutics. Major structural biology efforts on these proteins have already provided essential insights into the mode of action of the virus, as well as avenues for structure-based drug design. However, not all of the SARS-CoV-2 proteins, or regions thereof, have a well-defined three-dimensional structure, and as such might exhibit ambiguous, dynamic behaviour that is not evident from static structure representations, nor from molecular dynamics simulations using these structures. Main We present a website (https://bio2byte.be/sars2/) that provides protein sequence-based predictions of the backbone and side-chain dynamics and conformational propensities of these proteins, as well as derived early folding, disorder, β-sheet aggregation, protein-protein interaction and epitope propensities. These predictions attempt to capture the inherent biophysical propensities encoded in the sequence, rather than context-dependent behaviour such as the final folded state. In addition, we provide the biophysical variation that is observed in homologous proteins, which gives an indication of the limits of their functionally relevant biophysical behaviour. Conclusion The https://bio2byte.be/sars2/ website provides a range of protein sequence-based predictions for 27 SARS-CoV-2 proteins, enabling researchers to form hypotheses about their possible functional modes of action.

Immunogold localization of callose and other plant cell wall components in soybean roots infected with the oomycete Phytophthora sojae

1997 ◽  
Vol 75 (9) ◽  
pp. 1509-1517 ◽  
Author(s):  
K. Enkerli ◽  
C. W. Mims ◽  
M. G. Hahn
Keyword(s):  
Plasma Membrane ◽  
Plant Cell Wall ◽  
Arabinogalactan Proteins ◽  
Phytophthora Sojae ◽  
Arabinogalactan Protein ◽  
Electron Microscopic ◽  
Rhamnogalacturonan I ◽  
Host Plasma Membrane ◽  
Wall Appositions ◽  
Extrahaustorial Matrix

Immunolabeling and transmission electron microscopic techniques were used to investigate the chemical nature of wall appositions in roots of susceptible and resistant soybean plants inoculated with Phytophthora sojae race 2. The extrahaustorial matrix associated with the haustorium of Phytophthora sojae also was examined. Antibodies against (1 → 3)-β-glucan, a terminal α-fucosyl-containing epitope present in xyloglucan and rhamnogalacturonan I, and an arabinosylated (1 → 6)-β-galactan epitope present in arabinogalactan proteins were used. (1 → 3)-β-Glucan (callose), xyloglucan, and arabinogalactan proteins were found to be localized in all wall appositions regardless of how long after inoculation the appositions developed or whether plants were susceptible or resistant to Phytophthora sojae. (1 → 3)-β-Glucan also was found in fungal walls and at host cell plasmodesmata. None of the four antibodies labeled the extrahaustorial matrix. The antibody against arabinogalactan protein recognized the host plasma membrane, but not the invaginated host plasma membrane associated with the extrahaustorial matrix. This result indicates that the properties or the composition of the host plasma membrane may change locally once it becomes an extrahaustorial membrane. Key words: Phytophthora sojae, Glycine max, callose, immunolabeling, wall appositions, papillae.

scholarly journals Structural Basis for Env Incorporation into HIV-1 Particles

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 114
Author(s):  
R. Elliot Murphy ◽  
Alexandra B. Samal ◽  
Gunnar Eastep ◽  
Ruba H. Ghanam ◽  
Peter E. Prevelige ◽  
...  
Keyword(s):  
Structural Characterization ◽  
Late Phase ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Membrane Interactions ◽  
Env Protein ◽  
Gag Assembly ◽  
Hiv 1

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.

scholarly journals Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis

2014 ◽  
Vol 70 (5) ◽  
pp. 578-582 ◽  
Author(s):  
Marco Kloos ◽  
Antje Brüser ◽  
Jürgen Kirchberger ◽  
Torsten Schöneberg ◽  
Norbert Sträter
Keyword(s):  
Three Dimensional ◽  
Crystallographic Analysis ◽  
Human Muscle ◽  
Yeast Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Diffusion Method ◽  
Rabbit Muscle ◽  
Main Regulator ◽  
Purification Protocol

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space groupP6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and groupB-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.

scholarly journals The three-dimensional structure of the cellobiohydrolase Cel7A fromAspergillus fumigatusat 1.5 Å resolution

2015 ◽  
Vol 71 (1) ◽  
pp. 114-120 ◽  
Author(s):  
Olga V. Moroz ◽  
Michelle Maranta ◽  
Tarana Shaghasi ◽  
Paul V. Harris ◽  
Keith S. Wilson ◽  
...  
Keyword(s):  
Enzymatic Degradation ◽  
Plant Cell Wall ◽  
Three Dimensional ◽  
Biofuel Production ◽  
Industrial Processes ◽  
Dimensional Structure ◽  
Carbohydrate Active Enzymes ◽  
Cazy Database ◽  
Generation Biofuel ◽  
Insight Into

The enzymatic degradation of plant cell-wall cellulose is central to many industrial processes, including second-generation biofuel production. Key players in this deconstruction are the fungal cellobiohydrolases (CBHs), notably those from family GH7 of the carbohydrate-active enzymes (CAZY) database, which are generally known as CBHI enzymes. Here, three-dimensional structures are reported of theAspergillus fumigatusCBHI Cel7A solved in uncomplexed and disaccharide-bound forms at resolutions of 1.8 and 1.5 Å, respectively. The product complex with a disaccharide in the +1 and +2 subsites adds to the growing three-dimensional insight into this family of industrially relevant biocatalysts.

scholarly journals Prediction of fluorophore brightness in designed mini fluorescence activating proteins

2021 ◽  
Author(s):  
Emma R. Hostetter ◽  
Jeffrey R. Keyes ◽  
Ivy Poon ◽  
Justin P. Nguyen ◽  
Jacob Nite ◽  
...  
Keyword(s):  
Protein Function ◽  
De Novo ◽  
Energy Landscape ◽  
Three Dimensional ◽  
Computational Design ◽  
Dimensional Structure ◽  
Bond Rotation ◽  
Beta Barrel ◽  
Angle Rotation ◽  
Dynamics Simulations

The de novo computational design of proteins with predefined three-dimensional structure is becoming much more routine due to advancements both in force fields and algorithms. However, creating designs with functions beyond folding is more challenging. In that regard, the recent design of small beta barrel proteins that activate the fluorescence of an exogenous small molecule chromophore (DFHBI) is noteworthy. These proteins, termed mini Fluorescence Activating Proteins (mFAPs), have been shown increase the brightness of the chromophore more than 100-fold upon binding to the designed ligand pocket. The design process created a large library of variants with different brightness levels but gave no rational explanation for why one variant was brighter than another. Here we use quantum mechanics and molecular dynamics simulations to investigate how molecular flexibility in the ground and excited states influences brightness. We show that the ability of the protein to resist dihedral angle rotation of the chromophore is critical for predicting brightness. Our simulations suggest that the mFAP/DFHBI complex has a rough energy landscape, requiring extensive ground-state sampling to achieve converged predictions of excited-state kinetics. While computationally demanding, this roughness suggests that mFAP protein function can be enhanced by reshaping the energy landscape towards states that better resist DFHBI bond rotation.

scholarly journals Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

2021 ◽  
pp. mbc.E20-12-0806
Author(s):  
Yanhe Zhao ◽  
Justine Pinskey ◽  
Jianfeng Lin ◽  
Weining Yin ◽  
Patrick R. Sears ◽  
...  
Keyword(s):  
Primary Ciliary Dyskinesia ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Tract Infections ◽  
Ciliary Dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A ( RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native, three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected non-invasively from a PCD patient. Using cryo-electron tomography and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. [Media: see text]

scholarly journals Amphotericin B Assembles into Seven-Molecule Ion Channels in Membrane Domain

2021 ◽  
Author(s):  
Yuichi Umegawa ◽  
Tomoya Yamamoto ◽  
Mayank Dixit ◽  
Kosuke Funahashi ◽  
Sangjae Seo ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Resonance Spectroscopy ◽  
Selective Toxicity ◽  
Drug Molecules ◽  
Conductive Channel ◽  
High Resolution Structure ◽  
Dynamics Simulations ◽  
Resolution Structure

Amphotericin B, a long-used antifungal drug, forms fungicidal ion-permeable channels across cell membranes. Using solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we experimentally elucidated the three-dimensional structure of the molecular assemblies formed by this drug in membranes in the presence of the fungal sterol, ergosterol. A stable assembly of seven drug molecules was observed to form an ion conductive channel. The structure somewhat resembled the upper half of the barrel-stave model proposed in the 1970s but different substantially in the number of molecules and their arrangement. Based on the structure obtained, the aggregation of the channel assemblies in membranes was investigated and a mechanism was proposed in which complexation with ergosterol stabilizes the drug’s assemblies, leading to their aggregation, and in turn enhancing channel activity. The high-resolution structure is consistent with many previous findings, including structure-activity relationships of the drug, and the channel aggregation provides a more reasonable explanation for the selective toxicity of this drug to fungi.

scholarly journals Integrating NMR and simulations reveals motions in the UUCG tetraloop

2020 ◽  
Vol 48 (11) ◽  
pp. 5839-5848 ◽  
Author(s):  
Sandro Bottaro ◽  
Parker J Nichols ◽  
Beat Vögeli ◽  
Michele Parrinello ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Experimental Data ◽  
Three Dimensional ◽  
Machine Learning Techniques ◽  
Dimensional Structure ◽  
Stable Conformation ◽  
Stem Loop ◽  
Loop Region ◽  
Simple Model System ◽  
Nuclear Overhauser ◽  
Dynamics Simulations

Abstract We provide an atomic-level description of the structure and dynamics of the UUCG RNA stem–loop by combining molecular dynamics simulations with experimental data. The integration of simulations with exact nuclear Overhauser enhancements data allowed us to characterize two distinct states of this molecule. The most stable conformation corresponds to the consensus three-dimensional structure. The second state is characterized by the absence of the peculiar non-Watson–Crick interactions in the loop region. By using machine learning techniques we identify a set of experimental measurements that are most sensitive to the presence of non-native states. We find that although our MD ensemble, as well as the consensus UUCG tetraloop structures, are in good agreement with experiments, there are remaining discrepancies. Together, our results show that (i) the MD simulation overstabilize a non-native loop conformation, (ii) eNOE data support its presence with a population of ≈10% and (iii) the structural interpretation of experimental data for dynamic RNAs is highly complex, even for a simple model system such as the UUCG tetraloop.

scholarly journals Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase

2016 ◽  
Vol 198 (10) ◽  
pp. 1499-1512 ◽  
Author(s):  
Sonali Dhindwal ◽  
Leticia Gomez-Gil ◽  
David B. Neau ◽  
Thi Thanh My Pham ◽  
Michel Sylvestre ◽  
...  
Keyword(s):  
Polychlorinated Biphenyl ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Degradation Pathway ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Biphenyl Dioxygenase ◽  
Pcb Congeners ◽  
Biphenyl Degradation ◽  
Insight Into

ABSTRACTBiphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAEII9, a variant of BphAELB400in which a seven-residue segment,335TFNNIRI341, has been replaced by the corresponding segment of BphAEB356,333GINTIRT339, transforms a broader range of PCB congeners than does either BphAELB400or BphAEB356, including 2,6-dichlorobiphenyl, 3,3′-dichlorobiphenyl, 4,4′-dichlorobiphenyl, and 2,3,4′-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAEII9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAELB400reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAEII9that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAEII9transformed 2,3,4′-trichlorobiphenyl and 2,2′,5,5′-tetrachlorobiphenyl more efficiently than did BphAELB400and BphAEB356. BphAEII9also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparentkcat/Kmof 2.2 ± 0.5 mM−1s−1, versus 0.9 ± 0.5 mM−1s−1for BphAEB356). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes.IMPORTANCEBiphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. The structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.

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