scholarly journals The Protein’s Role in Substrate Positioning and Reactivity for Biosynthetic Enzyme Complexes: the Case of SyrB2/SyrB1

2018 ◽  
Author(s):  
Rimsha Mehmood ◽  
Helena W. Qi ◽  
Adam H. Steeves ◽  
Heather Kulik
Keyword(s):  
Free Energy ◽  
Large Scale ◽  
Structural Information ◽  
Acyl Carrier Protein ◽  
Protein Complexes ◽  
Heme Iron ◽  
Free Energy Barrier ◽  
Chemical Transformations ◽  
Biosynthetic Enzyme ◽  
Enzyme Complexes

Biosynthetic enzyme complexes selectively catalyze challenging chemical transformations, including alkane functionalization (e.g., halogenation of threonine, Thr, by non-heme iron SyrB2). However, the role of complex formation in enabling reactivity and guiding selectivity is poorly understood, owing to the challenges associated with obtaining detailed structural information of the dynamically associating protein complexes. Combining over 10 ms of classical molecular dynamics of SyrB2 and the acyl carrier protein SyrB1 with large-scale QM/MM simulation, we investigate the substrate–protein and protein–protein dynamics that give rise to experimentally observed substrate positioning and reactivity trends. We confirm the presence of a hypothesized substrate-delivery channel in SyrB2 through free energy simulations that show channel opening with a low free energy barrier. We identify stabilizing interactions at the SyrB2/SyrB1 interface that are compatible with phosphopantatheine (PPant) delivery of substrate to SyrB2. By sampling metal–substrate distances observed in experimental spectroscopy of native SyrB2/SyrB1-PPant-<i>S</i>-Thr and non-native substrates, we characterize essential protein–substrate interactions that are responsible for substrate positioning, and thus, reactivity. We observe the hydroxyl sidechain and terminal amine of the native Thr substrate to form cooperative hydrogen bonds with a single N123 residue in SyrB2. In comparison, non-native substrates that lack the hydroxyl interact more flexibly with the protein and therefore can orient closer to the Fe center, explaining their preferential hydroxylation and higher turnover frequencies.

scholarly journals The Protein’s Role in Substrate Positioning and Reactivity for Biosynthetic Enzyme Complexes: the Case of SyrB2/SyrB1

2018 ◽  
Author(s):  
Rimsha Mehmood ◽  
Helena W. Qi ◽  
Adam H. Steeves ◽  
Heather Kulik
Keyword(s):  
Free Energy ◽  
Large Scale ◽  
Structural Information ◽  
Acyl Carrier Protein ◽  
Protein Complexes ◽  
Heme Iron ◽  
Free Energy Barrier ◽  
Chemical Transformations ◽  
Biosynthetic Enzyme ◽  
Enzyme Complexes

Biosynthetic enzyme complexes selectively catalyze challenging chemical transformations, including alkane functionalization (e.g., halogenation of threonine, Thr, by non-heme iron SyrB2). However, the role of complex formation in enabling reactivity and guiding selectivity is poorly understood, owing to the challenges associated with obtaining detailed structural information of the dynamically associating protein complexes. Combining over 10 ms of classical molecular dynamics of SyrB2 and the acyl carrier protein SyrB1 with large-scale QM/MM simulation, we investigate the substrate–protein and protein–protein dynamics that give rise to experimentally observed substrate positioning and reactivity trends. We confirm the presence of a hypothesized substrate-delivery channel in SyrB2 through free energy simulations that show channel opening with a low free energy barrier. We identify stabilizing interactions at the SyrB2/SyrB1 interface that are compatible with phosphopantatheine (PPant) delivery of substrate to SyrB2. By sampling metal–substrate distances observed in experimental spectroscopy of native SyrB2/SyrB1-PPant-<i>S</i>-Thr and non-native substrates, we characterize essential protein–substrate interactions that are responsible for substrate positioning, and thus, reactivity. We observe the hydroxyl sidechain and terminal amine of the native Thr substrate to form cooperative hydrogen bonds with a single N123 residue in SyrB2. In comparison, non-native substrates that lack the hydroxyl interact more flexibly with the protein and therefore can orient closer to the Fe center, explaining their preferential hydroxylation and higher turnover frequencies.

ProAffiMuSeq: sequence-based method to predict the binding free energy change of protein–protein complexes upon mutation using functional classification

2019 ◽  
Author(s):  
Sherlyn Jemimah ◽  
Masakazu Sekijima ◽  
M Michael Gromiha
Keyword(s):  
Free Energy ◽  
Protein Interactions ◽  
Large Scale ◽  
Structural Information ◽  
Protein Complexes ◽  
Binding Free Energy ◽  
Complex Structure ◽  
External Validation ◽  
Functional Class ◽  
Supplementary Information

Abstract Motivation Protein–protein interactions are essential for the cell and mediate various functions. However, mutations can disrupt these interactions and may cause diseases. Currently available computational methods require a complex structure as input for predicting the change in binding affinity. Further, they have not included the functional class information for the protein–protein complex. To address this, we have developed a method, ProAffiMuSeq, which predicts the change in binding free energy using sequence-based features and functional class. Results Our method shows an average correlation between predicted and experimentally determined ΔΔG of 0.73 and mean absolute error (MAE) of 0.86 kcal/mol in 10-fold cross-validation and correlation of 0.75 with MAE of 0.94 kcal/mol in the test dataset. ProAffiMuSeq was also tested on an external validation set and showed results comparable to structure-based methods. Our method can be used for large-scale analysis of disease-causing mutations in protein–protein complexes without structural information. Availability and implementation Users can access the method at https://web.iitm.ac.in/bioinfo2/proaffimuseq/. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals The Dynamics of Subunit Rotation in a Eukaryotic Ribosome

Biophysica ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 204-221
Author(s):  
Frederico Campos Freitas ◽  
Gabriele Fuchs ◽  
Ronaldo Junio de Oliveira ◽  
Paul Charles Whitford
Keyword(s):  
Free Energy ◽  
Large Scale ◽  
Small Subunit ◽  
Free Energy Barrier ◽  
Temperature Dependent ◽  
Molecular Flexibility ◽  
Subunit Rotation ◽  
Biological Kinetics ◽  
Rate Limiting ◽  
Reversible Rotation

Protein synthesis by the ribosome is coordinated by an intricate series of large-scale conformational rearrangements. Structural studies can provide information about long-lived states, however biological kinetics are controlled by the intervening free-energy barriers. While there has been progress describing the energy landscapes of bacterial ribosomes, very little is known about the energetics of large-scale rearrangements in eukaryotic systems. To address this topic, we constructed an all-atom model with simplified energetics and performed simulations of subunit rotation in the yeast ribosome. In these simulations, the small subunit (SSU; ∼1 MDa) undergoes spontaneous and reversible rotation events (∼8∘). By enabling the simulation of this rearrangement under equilibrium conditions, these calculations provide initial insights into the molecular factors that control dynamics in eukaryotic ribosomes. Through this, we are able to identify specific inter-subunit interactions that have a pronounced influence on the rate-limiting free-energy barrier. We also show that, as a result of changes in molecular flexibility, the thermodynamic balance between the rotated and unrotated states is temperature-dependent. This effect may be interpreted in terms of differential molecular flexibility within the rotated and unrotated states. Together, these calculations provide a foundation, upon which the field may begin to dissect the energetics of these complex molecular machines.

scholarly journals Large-scale binding affinity calculations on commodity compute clouds

2020 ◽  
Vol 10 (6) ◽  
pp. 20190133
Author(s):  
S. J. Zasada ◽  
D. W. Wright ◽  
P. V. Coveney
Keyword(s):  
Free Energy ◽  
Binding Affinity ◽  
Large Scale ◽  
Model Building ◽  
Structural Information ◽  
Md Simulations ◽  
Free Energy Calculations ◽  
Binding Affinities ◽  
Software Infrastructure ◽  
Computing Platforms

In recent years, it has become possible to calculate binding affinities of compounds bound to proteins via rapid, accurate, precise and reproducible free energy calculations. This is imperative in drug discovery as well as personalized medicine. This approach is based on molecular dynamics (MD) simulations and draws on sequence and structural information of the protein and compound concerned. Free energies are determined by ensemble averages of many MD replicas, each of which requires hundreds of cores and/or GPU accelerators, which are now available on commodity cloud computing platforms; there are also requirements for initial model building and subsequent data analysis stages. To automate the process, we have developed a workflow known as the binding affinity calculator. In this paper, we focus on the software infrastructure and interfaces that we have developed to automate the overall workflow and execute it on commodity cloud platforms, in order to reliably predict their binding affinities on time scales relevant to the domains of application, and illustrate its application to two free energy methods.

scholarly journals The dynamics of subunit rotation in a eukaryotic ribosome

2021 ◽  
Author(s):  
Frederico Campos Freitas ◽  
Gabriele Fuchs ◽  
Ronaldo Junio de Oliveira ◽  
Paul Charles Whitford
Keyword(s):  
Free Energy ◽  
Large Scale ◽  
Small Subunit ◽  
Free Energy Barrier ◽  
Temperature Dependent ◽  
Molecular Flexibility ◽  
Subunit Rotation ◽  
Conformational Rearrangements ◽  
Biological Kinetics ◽  
Rate Limiting

AbstractProtein synthesis by the ribosome is coordinated by an intricate series of large-scale conformational rearrangements. Structural studies can provide information about long-lived states, however biological kinetics are controlled by the intervening free-energy barriers. While there has been progress describing the energy landscapes of bacterial ribosomes, very little is known about the energetics of large-scale rearrangements in eukaryotic systems. To address this topic, we constructed an all-atom model with simplified energetics and performed simulations of subunit rotation in the yeast ribosome. In these simulations, the small subunit (SSU; ~1MDa) undergoes spontaneous and reversible rotations (~ 8°). By enabling the simulation of this rearrangement under equilibrium conditions, these calculations provide initial insights into the molecular factors that control dynamics in eukaryotic ribosomes. Through this, we are able to identify specific inter-subunit interactions that have a pronounced influence on the rate-limiting free-energy barrier. We also show that, as a result of changes in molecular flexibility, the thermodynamic balance between the rotated and unrotated states is temperature-dependent. This effect may be interpreted in terms of differential molecular flexibility within the rotated and unrotated states. Together, these calculations provide a foundation, upon which the field may begin to dissect the energetics of these complex molecular machines.

scholarly journals The nematic-isotropic transition of the Lebwohl–Lasher model revisited

2021 ◽  
Vol 379 (2201) ◽  
pp. 20200117 ◽  
Author(s):  
Gregor Skačej ◽  
Claudio Zannoni
Keyword(s):  
Monte Carlo ◽  
Free Energy ◽  
Transition Temperature ◽  
Energy Barrier ◽  
Nematic Phase ◽  
Large Scale ◽  
Free Energy Barrier ◽  
Complex Materials ◽  
Theme Issue ◽  
Mathematical Design

We revisited the nematic-isotropic (NI) transition of the Lebwohl–Lasher lattice model with a detailed investigation of samples containing 200 × 200 × 200 particles. The large-scale Monte Carlo (MC) simulations involved were carried out following the standard Metropolis, as well as the cluster MC Wolff algorithms. A notable free-energy barrier was observed between the isotropic and nematic phase, leading to long-lived metastable states and hysteresis. We provide an improved estimate of the nematic-isotropic transition temperature T NI , of the supercooling and superheating temperatures, of the temperature of divergence of pretransitional effects T C ∗ as well as an analysis of the size distribution of the ordered domains above T NI , contributing to a better understanding of this transition of key importance for liquid crystals. This article is part of the theme issue ‘Topics in mathematical design of complex materials’.

scholarly journals Reliable identification of protein-protein interactions by crosslinking mass spectrometry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Swantje Lenz ◽  
Ludwig R. Sinn ◽  
Francis J. O’Reilly ◽  
Lutz Fischer ◽  
Fritz Wegner ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Large Scale ◽  
Structural Information ◽  
Protein Complexes ◽  
Interaction Network ◽  
Protein Protein Interactions ◽  
False Discovery Rates ◽  
Large Scale Analysis ◽  
Exit Tunnel

AbstractProtein-protein interactions govern most cellular pathways and processes, and multiple technologies have emerged to systematically map them. Assessing the error of interaction networks has been a challenge. Crosslinking mass spectrometry is currently widening its scope from structural analyses of purified multi-protein complexes towards systems-wide analyses of protein-protein interactions (PPIs). Using a carefully controlled large-scale analysis of Escherichia coli cell lysate, we demonstrate that false-discovery rates (FDR) for PPIs identified by crosslinking mass spectrometry can be reliably estimated. We present an interaction network comprising 590 PPIs at 1% decoy-based PPI-FDR. The structural information included in this network localises the binding site of the hitherto uncharacterised protein YacL to near the DNA exit tunnel on the RNA polymerase.

Large-Scale Assessment of Binding Free Energy Calculations in Active Drug Discovery Projects

2020 ◽  
Author(s):  
Christina Schindler ◽  
Hannah Baumann ◽  
Andreas Blum ◽  
Dietrich Böse ◽  
Hans-Peter Buchstaller ◽  
...  
Keyword(s):  
Free Energy ◽  
Drug Discovery ◽  
Large Scale ◽  
Active Drug ◽  
Binding Free Energy ◽  
Free Energy Calculations ◽  
Energy Calculation ◽  
Large Scale Assessment ◽  
New Public ◽  
Binding Free Energy Calculations

Here we present an evaluation of the binding affinity prediction accuracy of the free energy calculation method FEP+ on internal active drug discovery projects and on a large new public benchmark set.<br>

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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