scholarly journals Estimation of effective concentrations enforced by complex linker architectures using conformational ensembles.

2021 ◽  
Author(s):  
Magnus Kjaergaard
Keyword(s):  
Effective Concentration ◽  
Conformational Ensemble ◽  
Protein Assemblies ◽  
Conformational Ensembles ◽  
Excluded Volume Effects ◽  
Interaction Partners ◽  
End Distance ◽  
Molecular Context ◽  
Polymer Models ◽  
General Method

Proteins and protein assemblies often tether interaction partners to strengthen interactions, to regulate activity through auto-inhibition or -activation, or to boost enzyme catalysis. Tethered reactions are regulated by the architecture of tether, which define an effective concentration of the interactors. Effective concentrations can be estimated theoretically for simple linkers via polymer models, but there is currently no general method for estimating effective concentrations for complex linker architectures consisting of both flexible and folded domains. We describe how effective concentrations can be estimated computationally for any protein linker architecture by defining a realistic conformational ensemble. We benchmark against prediction from a worm-like chain and values measured by competition experiments, and find minor differences likely due to excluded volume effects. Systematic variation of the properties of flexible and folded segments show that the effective concentration is mainly determined by the combination of the total length of flexible segments and the distance between termini of the folded domains. We show that a folded domain in a disordered linker can increase the effective concentration beyond what can be achieved by a fully disordered linker by focusing the end-to-end distance at the appropriate spacing. This suggest that complex linker architecture may have advantages over simple flexible linker, and emphasize that annotation as a linker should depend on the molecular context.

scholarly journals Determination of Protein Structural Ensembles by Hybrid-Resolution SAXS Restrained Molecular Dynamics.

2019 ◽  
Author(s):  
Cristina Paissoni ◽  
Alexander Jussupow ◽  
Carlo Camilloni
Keyword(s):  
Dynamical Behavior ◽  
Computational Cost ◽  
Three Dimensional ◽  
Md Simulations ◽  
Coarse Grained ◽  
Conformational Ensemble ◽  
Biomolecular Systems ◽  
Conformational Ensembles ◽  
Independent Experimental Data

<div><div><div><p>SAXS experiments provide low-resolution but valuable information about the dynamics of biomolecular systems, which could be ideally integrated in MD simulations to accurately determine conformational ensembles of flexible proteins. The applicability of this strategy is hampered by the high computational cost required to calculate scattering intensities from three-dimensional structures. We previously presented a metainference-based hybrid resolution method that makes atomistic SAXS-restrained MD simulation feasible by adopting a coarse-grained approach to efficiently back-calculate scattering intensities; here, we extend this technique, applying it in the framework of multiple-replica simulations with the aim to investigate the dynamical behavior of flexible biomolecules. The efficacy of the method is assessed on the K63-diubiquitin multi-domain protein, showing that inclusion of SAXS-restraints is effective in generating reliable and heterogenous conformational ensemble, also improving the agreement with independent experimental data.</p></div></div></div>

scholarly journals How to Determine Accurate Conformational Ensembles by Metadynamics Metainference: A Chignolin Study Case

2021 ◽  
Vol 8 ◽  
Author(s):  
Cristina Paissoni ◽  
Carlo Camilloni
Keyword(s):  
Experimental Data ◽  
Molecular Dynamics ◽  
Experimental Information ◽  
Conformational Ensemble ◽  
Enhanced Sampling ◽  
Conformational Ensembles ◽  
Statistical Precision ◽  
Minimum Number ◽  
Study Case ◽  
Dynamics Simulations

The reliability and usefulness of molecular dynamics simulations of equilibrium processes rests on their statistical precision and their capability to generate conformational ensembles in agreement with available experimental knowledge. Metadynamics Metainference (M&amp;M), coupling molecular dynamics with the enhanced sampling ability of Metadynamics and with the ability to integrate experimental information of Metainference, can in principle achieve both goals. Here we show that three different Metadynamics setups provide converged estimate of the populations of the three-states populated by a model peptide. Errors are estimated correctly by block averaging, but higher precision is obtained by performing independent replicates. One effect of Metadynamics is that of dramatically decreasing the number of effective frames resulting from the simulations and this is relevant for M&amp;M where the number of replicas should be large enough to capture the conformational heterogeneity behind the experimental data. Our simulations allow also us to propose that monitoring the relative error associated with conformational averaging can help to determine the minimum number of replicas to be simulated in the context of M&amp;M simulations. Altogether our data provides useful indication on how to generate sound conformational ensemble in agreement with experimental data.

scholarly journals Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Aron Broom ◽  
Rojo V. Rakotoharisoa ◽  
Michael C. Thompson ◽  
Niayesh Zarifi ◽  
Erin Nguyen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Protein Design ◽  
Active Site ◽  
Room Temperature ◽  
De Novo ◽  
Conformational Ensemble ◽  
Enzyme Design ◽  
X Ray ◽  
Conformational Ensembles ◽  
X Ray Crystallography

Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

scholarly journals Determination of Protein Structural Ensembles by Hybrid-Resolution SAXS Restrained Molecular Dynamics.

2019 ◽  
Author(s):  
Cristina Paissoni ◽  
Alexander Jussupow ◽  
Carlo Camilloni
Keyword(s):  
Dynamical Behavior ◽  
Computational Cost ◽  
Three Dimensional ◽  
Md Simulations ◽  
Coarse Grained ◽  
Conformational Ensemble ◽  
Biomolecular Systems ◽  
Conformational Ensembles ◽  
Independent Experimental Data

<div><div><div><p>SAXS experiments provide low-resolution but valuable information about the dynamics of biomolecular systems, which could be ideally integrated into MD simulations to accurately determine conformational ensembles of flexible proteins. The applicability of this strategy is hampered by the high computational cost required to calculate scattering intensities from three-dimensional structures. We previously presented a hybrid resolution method that makes atomistic SAXS-restrained MD simulation feasible by adopting a coarse-grained approach to efficiently back-calculate scattering intensities; here, we extend this technique, applying it in the framework of metainference with the aim to investigate the dynamical behavior of flexible biomolecules. The efficacy of the method is assessed on the K63-diubiquitin, showing that the inclusion of SAXS-restraints is effective in generating a reliable conformational ensemble, improving the agreement with independent experimental data.</p></div></div></div>

scholarly journals Coarse-grain simulations on NMR conformational ensembles highlight functional residues in proteins

2019 ◽  
Author(s):  
Sophie Sacquin-Mora
Keyword(s):  
Experimental Data ◽  
Mechanical Properties ◽  
Small Molecules ◽  
Protein Function ◽  
Coarse Grain ◽  
Conformational Ensemble ◽  
Structural Variations ◽  
New Approach ◽  
Conformational Ensembles ◽  
Dynamics Simulations

AbstractDynamics are a key feature of protein function, and this is especially true of gating residues, which occupy cavity or tunnel lining positions in the protein structure, and will reversibly switch between open and closed conformations in order to control the diffusion of small molecules within a protein’s internal matrix. Earlier work on globins and hydrogenases have shown that these gating residues can be detected using a multiscale scheme combining all atom classic molecular dynamics simulations and coarse grain calculations of the resulting conformational ensemble mechanical properties. Here we show that the structural variations observed in the conformational ensembles produced by NMR spectroscopy experiments are sufficient to induce noticeable mechanical changes in a protein, which in turn can be used to identify residues important for function and forming a mechanical nucleus in the protein core. This new approach, which combines experimental data and rapid coarse-grain calculations and no longer needs to resort to time-consuming all-atom simulations, was successfully applied to five different protein families.

scholarly journals Determination of Protein Structural Ensembles by Hybrid-Resolution SAXS Restrained Molecular Dynamics.

2019 ◽  
Author(s):  
Cristina Paissoni ◽  
Alexander Jussupow ◽  
Carlo Camilloni
Keyword(s):  
Dynamical Behavior ◽  
Computational Cost ◽  
Three Dimensional ◽  
Md Simulations ◽  
Coarse Grained ◽  
Conformational Ensemble ◽  
Biomolecular Systems ◽  
Conformational Ensembles ◽  
Independent Experimental Data

<div><div><div><p>SAXS experiments provide low-resolution but valuable information about the dynamics of biomolecular systems, which could be ideally integrated into MD simulations to accurately determine conformational ensembles of flexible proteins. The applicability of this strategy is hampered by the high computational cost required to calculate scattering intensities from three-dimensional structures. We previously presented a hybrid resolution method that makes atomistic SAXS-restrained MD simulation feasible by adopting a coarse-grained approach to efficiently back-calculate scattering intensities; here, we extend this technique, applying it in the framework of metainference with the aim to investigate the dynamical behavior of flexible biomolecules. The efficacy of the method is assessed on the K63-diubiquitin, showing that the inclusion of SAXS-restraints is effective in generating a reliable conformational ensemble, improving the agreement with independent experimental data.</p></div></div></div>

scholarly journals Coarse-grain simulations on NMR conformational ensembles highlight functional residues in proteins

2019 ◽  
Vol 16 (156) ◽  
pp. 20190075
Author(s):  
Sophie Sacquin-Mora
Keyword(s):  
Experimental Data ◽  
Mechanical Properties ◽  
Small Molecules ◽  
Protein Function ◽  
Coarse Grain ◽  
Conformational Ensemble ◽  
Structural Variations ◽  
New Approach ◽  
Conformational Ensembles ◽  
Dynamics Simulations

Dynamics are a key feature of protein function, and this is especially true of gating residues, which occupy cavity or tunnel lining positions in the protein structure, and will reversibly switch between open and closed conformations in order to control the diffusion of small molecules within a protein's internal matrix. Earlier work on globins and hydrogenases have shown that these gating residues can be detected using a multiscale scheme combining all-atom classic molecular dynamics simulations and coarse-grain calculations of the resulting conformational ensemble mechanical properties. Here, we show that the structural variations observed in the conformational ensembles produced by NMR spectroscopy experiments are sufficient to induce noticeable mechanical changes in a protein, which in turn can be used to identify residues important for function and forming a mechanical nucleus in the protein core. This new approach, which combines experimental data and rapid coarse-grain calculations and no longer needs to resort to time-consuming all-atom simulations, was successfully applied to five different protein families.

scholarly journals Properties of protein unfolded states suggest broad selection for expanded conformational ensembles

2020 ◽  
Vol 117 (38) ◽  
pp. 23356-23364 ◽  
Author(s):  
Micayla A. Bowman ◽  
Joshua A. Riback ◽  
Anabel Rodriguez ◽  
Hongyu Guo ◽  
Jun Li ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Protein Structures ◽  
Water Soluble ◽  
Disordered Proteins ◽  
Conformational Ensemble ◽  
Conformational Ensembles ◽  
Intrinsically Disordered ◽  
Sequence Patterns ◽  
Selection For

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein fromBordetella pertussis, as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

scholarly journals Computing, analyzing and comparing the radius of gyration and hydrodynamic radius in conformational ensembles of intrinsically disordered proteins

2019 ◽  
Author(s):  
Mustapha Carab Ahmed ◽  
Ramon Crehuet ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Hydrodynamic Radius ◽  
Intrinsically Disordered Proteins ◽  
Intrinsically Disordered Protein ◽  
Disordered Proteins ◽  
Radius Of Gyration ◽  
Conformational Ensemble ◽  
Conformational Ensembles ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Nmr Diffusion

AbstractThe level of compaction of an intrinsically disordered protein may affect both its physical and biological properties, and can be probed via different types of biophysical experiments. Small-angle X-ray scattering (SAXS) probe the radius of gyration (Rg) whereas pulsed-field-gradient nuclear magnetic resonance (NMR) diffusion, fluorescence correlation spectroscopy and dynamic light scattering experiments can be used to determine the hydrodynamic radius (Rh). Here we show how to calculate Rg and Rh from a computationally-generated conformational ensemble of an intrinsically disordered protein. We further describe how to use a Bayesian/Maximum Entropy procedure to integrate data from SAXS and NMR diffusion experiments, so as to derive conformational ensembles in agreement with those experiments.

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