scholarly journals Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP

2019 ◽  
Vol 151 (3) ◽  
pp. 381-394 ◽  
Author(s):  
Vanessa Leone ◽  
Izabela Waclawska ◽  
Katharina Kossmann ◽  
Caroline Koshy ◽  
Monika Sharma ◽  
...  
Keyword(s):  
Spectroscopic Data ◽  
Double Resonance ◽  
Structural Data ◽  
Spin Labels ◽  
Conformational Ensemble ◽  
Spectroscopic Techniques ◽  
Coupled Transport ◽  
X Ray Crystallography ◽  
Simulation Techniques ◽  
Double Electron Electron Resonance

Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron–electron double resonance (PELDOR), also known as double electron–electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.

scholarly journals Mutation of outer-shell residues modulates metal ion co-ordination strength in a metalloenzyme

2010 ◽  
Vol 429 (2) ◽  
pp. 313-321 ◽  
Author(s):  
Jee-Loon Foo ◽  
Colin J. Jackson ◽  
Paul D. Carr ◽  
Hye-Kyung Kim ◽  
Gerhard Schenk ◽  
...  
Keyword(s):  
Metal Ion ◽  
Structural Data ◽  
Biochemical Properties ◽  
Outer Shell ◽  
Model Systems ◽  
Spectroscopic Techniques ◽  
Structural Determinants ◽  
Ion Specificity ◽  
X Ray Crystallography ◽  
Metal Ion Interactions

The metal ion co-ordination sites of many metalloproteins have been characterized by a variety of spectroscopic techniques and small-molecule model systems, revealing many important insights into the structural determinants of metal ion co-ordination. However, our understanding of this fundamentally and practically important phenomenon remains frustratingly simplistic; in many proteins it is essentially impossible to predict metal ion specificity and the effects of remote ‘outer-shell’ residues on metal ion co-ordination strength are also poorly defined. This is exemplified by our inability to explain why metalloenzymes with identical metal ion co-ordination spheres, such as the closely related orthologues of bacterial PTE (phosphotriesterase) from Agrobacterium radiobacter and Pseudomonas diminuta, display different metal ion specificity and co-ordination strength. In the present study, we present a series of PTE variants that all possess identical metal ion co-ordination spheres, yet display large differences in their metal ion co-ordination strength. Using measurement of the rates of metal ion dissociation from the active site alongside analysis of structural data obtained through X-ray crystallography, we show that ‘outer-shell’ residues provide essential support for the metal ion ligands, in effect buttressing them in their optimal orientation. Remote mutations appear to modulate metal ion interactions by increasing or decreasing the stabilizing effects of these networks. The present study therefore provides a description of how the greater protein fold can be modified to ‘tune’ the strength of metal ion co-ordination and metal ion specificity, as well as reinforcing the concept of proteins as ensembles of conformational states with unique structures and biochemical properties.

scholarly journals qFit 3: Protein and ligand multiconformer modeling for X-ray crystallographic and single-particle cryo-EM density maps

2020 ◽  
Author(s):  
Blake T. Riley ◽  
Stephanie A. Wankowicz ◽  
Saulo H. P. de Oliveira ◽  
Gydo C. P. van Zundert ◽  
Daniel Hogan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Structural Data ◽  
Conformational Ensemble ◽  
Distributed Application ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Formidable Challenge ◽  
Density Maps

AbstractNew X-ray crystallography and cryo-electron microscopy (cryo-EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open-source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0.

scholarly journals DEER and RIDME Measurements of the Nitroxide-Spin Labelled Copper-Bound Amine Oxidase Homodimer from Arthrobacter Globiformis

2021 ◽  
Author(s):  
Hannah Russell ◽  
Rachel Stewart ◽  
Christopher Prior ◽  
Vasily S. Oganesyan ◽  
Thembaninkosi G. Gaule ◽  
...  
Keyword(s):  
Dipolar Coupling ◽  
Amine Oxidase ◽  
Spin Labels ◽  
Arthrobacter Globiformis ◽  
Copper Amine Oxidase ◽  
Dipole Interactions ◽  
Nitroxide Spin Labels ◽  
Double Electron Electron Resonance ◽  
Dipolar Modulation ◽  
Paramagnetic Centres

AbstractIn the study of biological structures, pulse dipolar spectroscopy (PDS) is used to elucidate spin–spin distances at nanometre-scale by measuring dipole–dipole interactions between paramagnetic centres. The PDS methods of Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) are employed, and their results compared, for the measurement of the dipolar coupling between nitroxide spin labels and copper-II (Cu(II)) paramagnetic centres within the copper amine oxidase from Arthrobacter globiformis (AGAO). The distance distribution results obtained indicate that two distinct distances can be measured, with the longer of these at c.a. 5 nm. Conditions for optimising the RIDME experiment such that it may outperform DEER for these long distances are discussed. Modelling methods are used to show that the distances obtained after data analysis are consistent with the structure of AGAO.

scholarly journals Phenylhydrazone and Quinazoline Derivatives from the Cold-Seep-Derived Fungus Penicillium oxalicum

Marine Drugs ◽  
2020 ◽  
Vol 19 (1) ◽  
pp. 9
Author(s):  
Ya-Ping Liu ◽  
Sheng-Tao Fang ◽  
Zhen-Zhen Shi ◽  
Bin-Gui Wang ◽  
Xiao-Nian Li ◽  
...  
Keyword(s):  
Deep Sea ◽  
Spectroscopic Data ◽  
2D Nmr ◽  
Penicillium Oxalicum ◽  
Marine Phytoplankton ◽  
Cold Seep ◽  
Phytoplankton Species ◽  
X Ray ◽  
X Ray Crystallography ◽  
Quinazoline Derivatives

Three new phenylhydrazones, penoxahydrazones A–C (compounds 1–3), and two new quinazolines, penoxazolones A (compound 4) and B (compound 5), with unique linkages were isolated from the fungus Penicillium oxalicum obtained from the deep sea cold seep. Their structures and relative configurations were assigned by analysis of 1D/2D NMR and mass spectroscopic data, and the absolute configurations of 1, 4, and 5 were established on the basis of X-ray crystallography or ECD calculations. Compound 1 represents the first natural phenylhydrazone-bearing steroid, while compounds 2 and 3 are rarely occurring phenylhydrazone tautomers. Compounds 4 and 5 are enantiomers that feature quinazoline and cinnamic acid units. Some isolates exhibited inhibition of several marine phytoplankton species and marine-derived bacteria.

scholarly journals Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xuesong Wang ◽  
Willem Jespers ◽  
Rubén Prieto-Díaz ◽  
Maria Majellaro ◽  
Adriaan P. IJzerman ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Structural Data ◽  
Binding Mode ◽  
Selective Recognition ◽  
Chiral Hplc ◽  
Binding Assays ◽  
Structural Determinants ◽  
X Ray Crystallography ◽  
Adenosine Receptor Antagonist ◽  
Energy Perturbation

AbstractThe four adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A2BAR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A2A and A2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A2BAR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V2506.51 in A2BAR, which is a leucine in all other ARs including the closely related A2AAR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V6.51 mutant A2AAR receptor. Taken together, this study provides further insights in the binding mode of these A2BAR antagonists, paving the way for future ligand optimization.

scholarly journals 4-vinylpyridine derivatives: Protonation, methylation and silver(I) coordination chemistry

2021 ◽  
pp. 174751982198965
Author(s):  
Guoqi Zhang
Keyword(s):  
Mass Spectrometry ◽  
Schiff Base ◽  
Coordination Chemistry ◽  
Elemental Analysis ◽  
Silver Complex ◽  
Spectroscopic Techniques ◽  
X Ray ◽  
X Ray Crystallography ◽  
Pyridine Moiety ◽  
New Compounds

( E)-4-[2-(Pyridin-4-yl)vinyl]benzaldehyde, containing both a 4-vinylpyridine and an aldehyde functionality, is utilized to develop new, highly conjugated chalcone compounds and a bis-Schiff base azine compound. The chalcone-containing compounds are further explored for their protonation, methylation and silver(I) coordination chemistry using the pyridine moiety. In parallel, a cyano-containing analogue, ( E)-4-[2-(pyridin-4-yl)vinyl]benzonitrile is also synthesized and studied for its silver(I) coordination chemistry. These new compounds are fully characterized by mass spectrometry, elemental analysis and spectroscopic techniques. The methylated product of ( E)-1-(9-anthryl)-3-{4-[2-(pyridin-4-yl)vinyl]phenyl}prop-2-en-1-one and a silver complex of ( E)-4-[2-(pyridin-4-yl)vinyl]benzonitrile are structurally determined by X-ray crystallography.

scholarly journals Structure Solution of Nano-Crystalline Small Molecules Using MicroED and Solid-State NMR Dipolar-Based Experiments

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4652
Author(s):  
Nghia Tuan Duong ◽  
Yoshitaka Aoyama ◽  
Katsumi Kawamoto ◽  
Toshio Yamazaki ◽  
Yusuke Nishiyama
Keyword(s):  
Spin Dynamics ◽  
Double Resonance ◽  
Three Dimensional ◽  
Saturation Pulse ◽  
X Ray Crystallography ◽  
Nano Crystalline ◽  
Structure Solution ◽  
Validation Procedure ◽  
R Factors ◽  
Shift Calculation

Three-dimensional electron diffraction crystallography (microED) can solve structures of sub-micrometer crystals, which are too small for single crystal X-ray crystallography. However, R factors for the microED-based structures are generally high because of dynamic scattering. That means R factor may not be reliable provided that kinetic analysis is used. Consequently, there remains ambiguity to locate hydrogens and to assign nuclei with close atomic numbers, like carbon, nitrogen, and oxygen. Herein, we employed microED and ssNMR dipolar-based experiments together with spin dynamics numerical simulations. The NMR dipolar-based experiments were 1H-14N phase-modulated rotational-echo saturation-pulse double-resonance (PM-S-RESPDOR) and 1H-1H selective recoupling of proton (SERP) experiments. The former examined the dephasing effect of a specific 1H resonance under multiple 1H-14N dipolar couplings. The latter examined the selective polarization transfer between a 1H-1H pair. The structure was solved by microED and then validated by evaluating the agreement between experimental and calculated dipolar-based NMR results. As the measurements were performed on 1H and 14N, the method can be employed for natural abundance samples. Furthermore, the whole validation procedure was conducted at 293 K unlike widely used chemical shift calculation at 0 K using the GIPAW method. This combined method was demonstrated on monoclinic l-histidine.

scholarly journals Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

2018 ◽  
Vol 293 (47) ◽  
pp. 18296-18308 ◽  
Author(s):  
Chelsea Vickers ◽  
Feng Liu ◽  
Kento Abe ◽  
Orly Salama-Alber ◽  
Meredith Jenkins ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biological Activities ◽  
Bacterial Species ◽  
Structural Data ◽  
Side Chain ◽  
Glycoside Hydrolase Family ◽  
X Ray Crystallography ◽  
Catalytic Mechanisms ◽  
Thickening Agents ◽  
Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

Crotaleschenine, an Alkaloid of Crotalaria leschenaultii

1988 ◽  
Vol 41 (4) ◽  
pp. 429 ◽  
Author(s):  
LW Smith ◽  
JA Edgar ◽  
RI Willing ◽  
RW Gable ◽  
MF Mackay ◽  
...  
Keyword(s):  
Spectroscopic Data ◽  
X Ray ◽  
X Ray Crystallography

An alkaloid of Crotalaria leschenaultii DC., previously reported as crispatine and now named crotaleschenine, has been re-investigated and shown to be (7β,8α-H,12α,13α,14α)-12β-hydroxy-1,2-didehydrocrotalane-11,15-dione.1 Spectroscopic data are presented and the stereochemistry determined by X-ray crystallography. The esterifying acid of crotaleschenine is identical with that of retusine , which is thereby determined as (1a,7β,8α-H,12α,13α,14α)-12β- hydroxycrotalane-11,15-dione.

scholarly journals A Structural Overview of Vascular Endothelial Growth Factors Pharmacological Ligands: From Macromolecules to Designed Peptidomimetics

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6759
Author(s):  
Xiaoqing Ye ◽  
Jean-François Gaucher ◽  
Michel Vidal ◽  
Sylvain Broussy
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Unnatural Amino Acids ◽  
Structural Data ◽  
Age Related Macular Degeneration ◽  
Vascular Endothelial ◽  
Binding Modes ◽  
Therapeutic Applications ◽  
X Ray Crystallography ◽  
Age Related

The vascular endothelial growth factor (VEGF) family of cytokines plays a key role in vasculogenesis, angiogenesis, and lymphangiogenesis. VEGF-A is the main member of this family, alongside placental growth factor (PlGF), VEGF-B/C/D in mammals, and VEGF-E/F in other organisms. To study the activities of these growth factors under physiological and pathological conditions, resulting in therapeutic applications in cancer and age-related macular degeneration, blocking ligands have been developed. These have mostly been large biomolecules like antibodies. Ligands with high affinities, at least in the nanomolar range, and accurate structural data from X-ray crystallography and NMR spectroscopy have been described. They constitute the main focus of this overview, which evidences similarities and differences in their binding modes. For VEGF-A ligands, and to a limited extent also for PlGF, a transition is now observed towards developing smaller ligands like nanobodies and peptides. These include unnatural amino acids and chemical modifications for designed and improved properties, such as serum stability and greater affinity. However, this review also highlights the scarcity of such small molecular entities and the striking lack of small organic molecule ligands. It also shows the gap between the rather large array of ligands targeting VEGF-A and the general absence of ligands binding other VEGF members, besides some antibodies. Future developments in these directions are expected in the upcoming years, and the study of these growth factors and their promising therapeutic applications will be welcomed.

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