233 research outputs found
A correspondence between solution-state dynamics of an individual protein and the sequence and conformational diversity of its family.
Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure, dynamics and function. Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using "Backrub" motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures. Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance (NMR) Residual Dipolar Couplings (RDCs). Using this ensemble, we probe two proposed relationships between properties of protein ensembles: (i) a link between native-state dynamics and the conformational heterogeneity observed in crystal structures, and (ii) a relation between dynamics of an individual protein and the conformational variability explored by its natural family. We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs, encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family. Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution. More practically, the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics
A facile Oxygen-17 NMR Method to Determine Effective Viscosity in Dilute, Molecularly Crowded and Confined Aqueous Media
This NMR probe of water dynamics enables viscosity determination in concentrated and crowded solutions and allows quantifying internal fluidity within biological condensates
Interaction of Human Islet Amyloid Polypeptide with Model Membranes in the Presence of a Novel Oligomer Modulator “anle138B”
Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression
Parkinson's disease (PD) represents an increasing problem in society. The oligomerization of alpha-synuclein (alpha Syn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study alpha Syn-mediated toxic mechanisms, we established cell lines with inducible overexpression of different alpha Syn constructs: alpha Syn, alpha Syn coupled to the fluorescence protein Venus (alpha Syn-Venus), and alpha Syn coupled to the N-terminal or C-terminal part of Venus (V1S and SV2, respectively) for a bimolecular fluorescence complementation assay (BiFC). Inducibility was achieved by applying modified GAL4-UAS or Cre-loxP systems and addition of tebufenozide or 4-OH-tamoxifen, respectively. Expression constructs were stably integrated into the host genome of H4 neuroglioma cells by lentiviral transduction. We here demonstrate a detailed investigation of the expression characteristics of inducible H4 cells showing low background expression and high inducibility. We observed increased protein load and aggregation of alpha Syn upon incubation with DMSO and FeCl3 along with an increase in cytotoxicity. In summary, we present a system for the creation of inducibly alpha Syn-overexpressing cell lines holding high potential for the screening for modulators of alpha Syn aggregation and alpha Syn-mediated toxicity
Relative Configuration of Micrograms of Natural Compounds Using Proton Residual Chemical Shift Anisotropy
[Abstract] 3D molecular structure determination is a challenge for organic compounds or natural products available in minute amounts. Proton/proton and proton/carbon correlations yield the constitution. J couplings and NOEs oftentimes supported by one-bond 1H,13C residual dipolar couplings (RDCs) or by 13C residual chemical shift anisotropies (RCSAs) provide the relative configuration. However, these RDCs or carbon RCSAs rely on 1% natural abundance of 13C preventing their use for compounds available only in quantities of a few 10’s of µgs. By contrast, 1H RCSAs provide similar information on spatial orientation of structural moieties within a molecule, while using the abundant 1H spin. Herein, 1H RCSAs are accurately measured using constrained aligning gels or liquid crystals and applied to the 3D structural determination of molecules with varying complexities. Even more, deuterated alignment media allow the elucidation of the relative configuration of around 35 µg of a briarane compound isolated from Briareum asbestinum.This work was supported by the Max Planck Society and grew out of a collaboration in the context of the Forschergruppe (FOR 934) continued now by the DFG (Gr1211/19–1 and Re1007/9–1)/CAPES 418729698 project. N.N. gratefully acknowledges the financial support by SERB, New Delhi for ECR Grant with File No.: ECR/2017/001811. This work was also funded by grants RTI2018-093634-B-C22 from the State Agency for Research (AEI) of Spain, both co-funded by the FEDER Programme from the European Union, BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP) and GRC2018/039 and AgrupaciĂłn EstratĂ©gica CICA-INIBIC ED431E 2018/03 from Xunta de Galicia. C.J., J.R., and D.P.P. acknowledge CESGA for the computational support. J.C.F. acknowledges predoctoral research stay grant Inditex-UDC. D.P.P. received a fellowship from the program National Council of Science and Technology (CONACYT) of Mexico and the Secretariat of Research, Innovation and Higher Education (SIIES) of Yucatan (Mexico). We also thank Dr. G. Jithender Reddy for one isotropic measurement. We also thank Dr. Christian Schmidt for his cooperation in the manufacturing of micro stretching device. ANV thanks CNPq for a research fellowship and financial support M(426216/2018–0)German Research Foundation; Gr1211/19–1German Research Foundation; Re1007/9–1Brasil. Coordenação de Aperfeiçoamento de Pessoal de NĂvel Superior (CAPES); 418729698RepĂşblica de la India. Science and Engineering Research Board; ECR/2017/001811Xunta de Galicia; 0474_BLUEBIOLAB_1_EXunta de Galicia; GRC2018/039Xunta de Galicia; ED431E 2018/03Brasil. Conselho Nacional de Desenvolvimento CientĂfico e TecnolĂłgico; M(426216/2018–0
relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data
Nuclear magnetic resonance (NMR) is a powerful tool for observing the motion of biomolecules at the atomic level. One technique, the analysis of relaxation dispersion phenomenon, is highly suited for studying the kinetics and thermodynamics of biological processes. Built on top of the relax computational environment for NMR dynamics is a new dispersion analysis designed to be comprehensive, accurate and easy-to-use. The software supports more models, both numeric and analytic, than current solutions. An automated protocol, available for scripting and driving the graphical user interface (GUI), is designed to simplify the analysis of dispersion data for NMR spectroscopists. Decreases in optimization time are granted by parallelization for running on computer clusters and by skipping an initial grid search by using parameters from one solution as the starting point for another —using analytic model results for the numeric models, taking advantage of model nesting, and using averaged non-clustered results for the clustered analysis. Availability and implementation: The software relax is written in Python with C modules and is released under the GPLv3+ license. Source code and precompiled binaries for all major operating systems are available from http://www.nmr-relax.com. Contact: [email protected]
relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data
International audienceNuclear Magnetic Resonance (NMR) is a powerful tool for observing the motion of biomolecules at the atomic level. One technique, the analysis of relaxation dispersion phenomenon, is highly suited for studying the kinetics and thermodynamics of biological processes. Built on top of the relax computational environment for NMR dynamics is a new dispersion analysis designed to be comprehensive, accurate and easy to use. The software supports more models, both numeric and analytic, than current solutions. An automated protocol, available for scripting and driving the GUI, is designed to simplify the analysis of dispersion data for NMR spectroscopists. Decreases in optimisation time are granted by parallelisation for running on computer clusters and by skipping an initial grid search by using parameters from one solution as the starting point for another – using analytic model results for the numeric models, taking advantage of model nesting, and using averaged non-clustered results for the clustered analysis. Availability: The software relax is written in Python with C modules and is released under the GPLv3+ licence. Source code and precompiled binaries for all major operating systems are available from http://www.nmr-relax.com
Guiding protein-ligand docking with different experimental NMR-data
Today's scoring functions are one of the main reasons that state-of-the-art protein-ligand dockings fail in about 20 % to 40 % of the targets due to the sometimes severe approximations they make. However these approximations are necessary for performance reasons. One possibility to overcome these problems is the inclusion of additional, preferably experimental information in the docking process. Especially ligand-based NMR experiments that are far less demanding than the solution of the whole complex structure are helpful.Here we present the inclusion of three different types of NMR-data into the ChemPLP scoring function of our docking tool PLANTS. First, STD and intra-ligand trNOE spectra were used to obtain distant constraints between ligand and protein atoms. This approach proved beneficial for the docking of larger peptide ligands i. e. the epitope of MUC-1 glycoprotein to the SM3 antibody.In the second part the usefulness of INPHARMA data is shown by combinig a score, evaluating the agreement between simulated and measured INPHARMA spectra, with the PLANTS ChemPLP scoring function. First results from rescoring after local optimization of the poses and full docking experiments are shown
Interaction of Cu(i) with the Met-X3-Met motif of alpha-synuclein: binding ligands, affinity and structural features
The identity of the Cu(i) binding ligands at Met-X3-Met site of AcαS and its role into the affinity and structural properties of the interaction were elucidated by NMR spectroscopy. We provide evidence that the source of ligands for Cu(i) binding to the Met-X3-Met site comes from the N-terminal acetyl group and the Met-1, Asp-2 and Met-5 residues. From the study of site-directed mutants and synthetic peptide models of αS we demonstrated the critical role played by Met-1 and Met-5 residues on the binding affinity of the Cu(i) complex, acting as the main metal anchoring residues. While having a more modest impact in the affinity features of Cu(i) binding, as compared to the Met residues, the N-terminal acetyl group and Asp-2 are important in promoting local helical conformations, contributing to the stabilization of these structures by favoring Cu(i) binding.Fil: Gentile, Iñaki. Laboratorio Max Planck de BiologĂa Estructural, QuĂmica y BiofĂsica Molecular de Rosario; ArgentinaFil: Garro, Hugo Alejandro. Laboratorio Max Planck de BiologĂa Estructural, QuĂmica y BiofĂsica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - San Luis. Instituto de Investigaciones en TecnologĂa QuĂmica. Universidad Nacional de San Luis. Facultad de QuĂmica, BioquĂmica y Farmacia. Instituto de Investigaciones en TecnologĂa QuĂmica; ArgentinaFil: Delgado Ocaña, Susana. Laboratorio Max Planck de BiologĂa Estructural, QuĂmica y BiofĂsica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: González, Nazareno. Laboratorio Max Planck de BiologĂa Estructural, QuĂmica y BiofĂsica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Strohäker, Timo. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Schibich, Daniela. Laboratorio Max Planck de BiologĂa Estructural, QuĂmica y BiofĂsica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Quintanar, Liliana. Centro de InvestigaciĂłn y de Estudios; MĂ©xicoFil: Sambrotta, Luis Jorge. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Zweckstetter, Markus. Max Planck Institute For Biophysical Chemistry; Alemania. Deutsches Zentrum FĂĽr Neurodegenerative Erkrankungen; AlemaniaFil: Griesinger, Christian. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Menacho Márquez, Mauricio Ariel. Laboratorio Max Planck de BiologĂa Estructural, QuĂmica y BiofĂsica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico Tecnol.conicet - Rosario. Unidad de Direccion; ArgentinaFil: Fernandez, Claudio Oscar. Laboratorio Max Planck de BiologĂa Estructural, QuĂmica y BiofĂsica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentina. Max Planck Institute For Biophysical Chemistry; Alemani
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