Consensus model of a cyanobacterial light-dependent protochlorophyllide oxidoreductase in its pigment-free apo-form and photoactive ternary complex

Abstract Photosynthetic organisms employ two different enzymes for the reduction of the C17 = C18 double bond of protochlorophyllide (Pchlide), yielding the chlorophyll precursor chlorophyllide. First, a nitrogenase-like, light-independent (dark-operative) Pchlide oxidoreductase and secondly, a light-dependent Pchlide oxidoreductase (LPOR). For the latter enzyme, despite decades of research, no structural information is available. Here, we use protein structure modelling, molecular dynamics (MD) simulations combined with multi-wavelength analytical ultracentrifugation (MWA-AUC) and small angle X-ray scattering (SAXS) experiments to derive a consensus model of the LPOR apoprotein and the substrate/cofactor/LPOR ternary complex. MWA-AUC and SAXS experiments independently demonstrate that the apoprotein is monomeric, while ternary complex formation induces dimerization. SAXS-guided modelling studies provide a full-length model of the apoprotein and suggest a tentative mode of dimerization for the LPOR ternary complex, supported by published cross-link constraints. Our study provides a first impression of the LPOR structural organization.

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The solution structures of higher-order human telomere G-quadruplex multimers

Nucleic Acids Research ◽

10.1093/nar/gkaa1285 ◽

2021 ◽

Vol 49 (3) ◽

pp. 1749-1768

Author(s):

Robert C Monsen ◽

Srinivas Chakravarthy ◽

William L Dean ◽

Jonathan B Chaires ◽

John O Trent

Keyword(s):

Analytical Ultracentrifugation ◽

Persistence Length ◽

Md Simulations ◽

Size Exclusion ◽

Scattering Data ◽

Solution Structures ◽

X Ray Scattering ◽

G Quadruplex ◽

Dynamics Simulations ◽

Global Fitting

Abstract Human telomeres contain the repeat DNA sequence 5′-d(TTAGGG), with duplex regions that are several kilobases long terminating in a 3′ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate models proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5′-d(TTAGGG)n, with n = 8, 12 and 16, fold into multimeric structures containing the maximal number (2, 3 and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of ∼34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

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The solution structures of higher-order human telomere G-quadruplex multimers

10.1101/2020.11.13.382036 ◽

2020 ◽

Author(s):

Robert C. Monsen ◽

Srinivas Chakravarthy ◽

William L. Dean ◽

Jonathan B. Chaires ◽

John O. Trent

Keyword(s):

Analytical Ultracentrifugation ◽

Persistence Length ◽

Md Simulations ◽

Size Exclusion ◽

Scattering Data ◽

Solution Structures ◽

X Ray Scattering ◽

G Quadruplex ◽

Dynamics Simulations ◽

Global Fitting

ABSTRACTHuman telomeres contain the repeat DNA sequence 5’(TTAGGG), with duplex regions that are several kilobases long terminating in a 3’ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate modes proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5’(TTAGGG)n, with n=8, 12, and 16, fold into multimeric structures containing the maximal number (2, 3, and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of about 34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

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A Cross-Method Comparison of Sub-10 nm Nanoparticle Size Distribution

10.26434/chemrxiv.9891992.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ye Yang ◽

Suiyang Liao ◽

Zhi Luo ◽

Runzhang Qi ◽

Niamh Mac Fhionnlaoich ◽

...

Keyword(s):

Size Distribution ◽

Analytical Ultracentrifugation ◽

Statistical Significance ◽

Method Comparison ◽

Size Determination ◽

Size Dependent ◽

X Ray Scattering ◽

Transmission Electron ◽

Gold Nps ◽

Reliable Representation

Accurate nanoparticle (NP) size determination is essential across research domains, with many functions in nanoscience and biomedical research being size-dependent. Although transmission electron microscopy (TEM) is capable of resolving a single NP down to the sub-nm scale, the reliable representation of entire populations is plagued by challenges in providing statistical significance, predominantly due to limited sample counts, suboptimal preparation procedures and operator bias during image acquisition and analysis. Meanwhile alternative techniques exist, but reliable implementation requires a detailed understanding of appendant limitations. Herein, conventional TEM is compared to the size determination of sub-10 nm gold NPs in solution by small-angle X-ray scattering and analytical ultracentrifugation. Form-free Monte Carlo fitting of scattering profiles offers access to a direct representation of the core size distribution while ultracentrifugation sedimentation velocity analysis provides information of the hydrodynamic size distribution. We report a comparison of these three methods in determining the size of quasi-monodisperse, polydisperse and bimodal gold nanoparticles of 2 – 7 nm and discuss advantages and limitations of each technique.

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Predicting the Structure and Dynamics of Membrane Protein GerAB from Bacillus subtilis

International Journal of Molecular Sciences ◽

10.3390/ijms22073793 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3793

Author(s):

Sophie Blinker ◽

Jocelyne Vreede ◽

Peter Setlow ◽

Stanley Brul

Keyword(s):

Bacillus Subtilis ◽

Membrane Protein ◽

Spore Germination ◽

Structural Information ◽

Transmembrane Protein ◽

Homology Modelling ◽

Water Channel ◽

Md Simulations ◽

Integral Membrane Protein ◽

Starting Point

Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore’s inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an α-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination.

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NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure–Activity Relationships

International Journal of Molecular Sciences ◽

10.3390/ijms22020880 ◽

2021 ◽

Vol 22 (2) ◽

pp. 880

Author(s):

Thomas Schmitz ◽

Ajay Abisheck Paul George ◽

Britta Nubbemeyer ◽

Charlotte A. Bäuml ◽

Torsten Steinmetzer ◽

...

Keyword(s):

Structural Information ◽

Coagulation Factor ◽

Md Simulations ◽

2D Nmr ◽

Label Free ◽

2D Nmr Spectroscopy ◽

Structure Information ◽

Structure Activity Relationships ◽

Structure Activity ◽

Blood Sucking

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1–Cys37) and the flexible C-terminal part (Arg38–Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure–activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.

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Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications

Biochemical Society Transactions ◽

10.1042/bst20150138 ◽

2015 ◽

Vol 43 (5) ◽

pp. 1023-1032 ◽

Cited By ~ 10

Author(s):

Thomas Stockner ◽

Anna Mullen ◽

Fraser MacMillan

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Abc Transporters ◽

Epr Spectroscopy ◽

Structural Information ◽

Dynamic Properties ◽

Molecular System ◽

Synergistic Effects ◽

Md Simulations ◽

Substrate Spectrum

ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented.

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Investigation of the structure of regulatory proteins interacting with glycosaminoglycans by combining NMR spectroscopy and molecular modeling – the beginning of a wonderful friendship

Biological Chemistry ◽

10.1515/hsz-2021-0119 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Georg Künze ◽

Daniel Huster ◽

Sergey A. Samsonov

Keyword(s):

Nmr Spectroscopy ◽

Structural Information ◽

Md Simulations ◽

Specific Protein ◽

Regulatory Proteins ◽

Structural Constraints ◽

Modeling Tools ◽

Nuclear Overhauser ◽

Nmr Methods ◽

High Flexibility

Abstract The interaction of regulatory proteins with extracellular matrix or cell surface-anchored glycosaminoglycans (GAGs) plays important roles in molecular recognition, wound healing, growth, inflammation and many other processes. In spite of their high biological relevance, protein-GAG complexes are significantly underrepresented in structural databases because standard tools for structure determination experience difficulties in studying these complexes. Co-crystallization with subsequent X-ray analysis is hampered by the high flexibility of GAGs. NMR spectroscopy experiences difficulties related to the periodic nature of the GAGs and the sparse proton network between protein and GAG with distances that typically exceed the detection limit of nuclear Overhauser enhancement spectroscopy. In contrast, computer modeling tools have advanced over the last years delivering specific protein-GAG docking approaches successfully complemented with molecular dynamics (MD)-based analysis. Especially the combination of NMR spectroscopy in solution providing sparse structural constraints with molecular docking and MD simulations represents a useful synergy of forces to describe the structure of protein-GAG complexes. Here we review recent methodological progress in this field and bring up examples where the combination of new NMR methods along with cutting-edge modeling has yielded detailed structural information on complexes of highly relevant cytokines with GAGs.

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SAXS Merge: an automated statistical method to merge SAXS profiles using Gaussian processes

Journal of Synchrotron Radiation ◽

10.1107/s1600577513030117 ◽

2013 ◽

Vol 21 (1) ◽

pp. 203-208 ◽

Cited By ~ 11

Author(s):

Yannick G. Spill ◽

Seung Joong Kim ◽

Dina Schneidman-Duhovny ◽

Daniel Russel ◽

Ben Webb ◽

...

Keyword(s):

Statistical Method ◽

High Throughput ◽

Gaussian Processes ◽

Small Angle ◽

Structural Information ◽

Linear Interpolation ◽

High Quality ◽

X Ray ◽

X Ray Scattering ◽

Specific Order

Small-angle X-ray scattering (SAXS) is an experimental technique that allows structural information on biomolecules in solution to be gathered. High-quality SAXS profiles have typically been obtained by manual merging of scattering profiles from different concentrations and exposure times. This procedure is very subjective and results vary from user to user. Up to now, no robust automatic procedure has been published to perform this step, preventing the application of SAXS to high-throughput projects. Here,SAXS Merge, a fully automated statistical method for merging SAXS profiles using Gaussian processes, is presented. This method requires only the buffer-subtracted SAXS profiles in a specific order. At the heart of its formulation is non-linear interpolation using Gaussian processes, which provides a statement of the problem that accounts for correlation in the data.

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Solution-Based Structural Analysis of the Decaheme Cytochrome, MtrA, by Small-Angle X-ray Scattering and Analytical Ultracentrifugation

The Journal of Physical Chemistry B ◽

10.1021/jp203603r ◽

2011 ◽

Vol 115 (38) ◽

pp. 11208-11214 ◽

Cited By ~ 27

Author(s):

Mackenzie A. Firer-Sherwood ◽

Nozomi Ando ◽

Catherine L. Drennan ◽

Sean J. Elliott

Keyword(s):

Structural Analysis ◽

Small Angle ◽

Analytical Ultracentrifugation ◽

X Ray ◽

X Ray Scattering ◽

Ray Scattering

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Molecular Dynamics Modellization and Simulation of Water Diffusion through Plant Cutin

Zeitschrift für Naturforschung C ◽

10.1515/znc-1999-1107 ◽

1999 ◽

Vol 54 (11) ◽

pp. 896-902 ◽

Cited By ~ 5

Author(s):

Antonio Matas ◽

Antonio Heredia

Keyword(s):

Experimental Data ◽

Molecular Dynamics ◽

Structural Information ◽

Md Simulations ◽

Water Molecules ◽

Plant Cuticle ◽

X Ray Diffraction ◽

Cross Link ◽

X Ray ◽

Good Agreement

Abstract A theoretical molecular modelling study has been conducted for cutin, the biopolyester that forms the main structural component of the plant cuticle. Molecular dynamics (MD) simulations, extended over several ten picoseconds, suggests that cutin is a moderately flexible netting with motional constraints mainly located at the cross-link sites of functional ester groups. This study also gives structural information essentially in accordance with previously reported experimental data, obtained from X -ray diffraction and nuclear magnetic resonance experiments. MD calculations were also performed to simulate the diffusion of water molecules through the cutin biopolymer. The theoretical analysis gives evidence that water permeation proceedes by a “hopping mechanism”. Coefficients for the diffusion of the water molecules in cutin were obtained from their mean-square displacements yielding values in good agreement with experimental data.

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