scholarly journals Toward computational design of protein crystals with improved resolution

2019 ◽  
Author(s):  
Jeliazko R. Jeliazkov ◽  
Aaron C. Robinson ◽  
Bertrand García-Moreno E. ◽  
James M. Berger ◽  
Jeffrey J. Gray
Keyword(s):  
Weak Interactions ◽  
Design Method ◽  
Single Point ◽  
Point Mutations ◽  
Computational Design ◽  
Data Bank ◽  
Protein Crystals ◽  
Single Individual ◽  
Space Group ◽  
Crystal Space

AbstractSubstantial advances have been made in the computational design of protein interfaces over the last 20 years. However, the interfaces targeted by design have typically been stable and high affinity. Here, we report the development of a generic computational design method to stabilize the weak interactions at crystallographic interfaces. Initially, we analyzed structures reported in the Protein Data Bank (PDB) to determine whether crystals with more stable interfaces result in higher resolution structures. We found that, for twenty-two variants of a single protein crystallized by a single individual, Rosetta score correlates with resolution. We next developed and tested a computational design protocol, seeking to identify point mutations that would improve resolution on a highly stable variant of staphylococcal nuclease (SNase Δ+PHS). Only one of eleven initial designs crystallized, forcing us to re-evaluate our strategy and base our designs on an ensemble of protein backbones. Using this strategy, four of the five designed proteins crystallized. Collecting diffraction data for multiple crystals per design and solving crystal structures, we found that designed crystals improved resolution modestly and in unpredictable ways, including altering crystal space group. Post-hoc, in silico analysis showed that crystal space groups could have been predicted for four of six variants (including WT), but that resolution did not correlate with interface stability, as it did in the preliminary results. Our results show that single point mutations can have significant effects on crystal resolution and space group, and that it is possible to computationally identify such mutations, suggesting a potential design strategy to generate high-resolution protein crystals from poorly diffracting ones.

scholarly journals Toward the computational design of protein crystals with improved resolution

2019 ◽  
Vol 75 (11) ◽  
pp. 1015-1027 ◽  
Author(s):  
Jeliazko R. Jeliazkov ◽  
Aaron C. Robinson ◽  
Bertrand García-Moreno E. ◽  
James M. Berger ◽  
Jeffrey J. Gray
Keyword(s):  
Weak Interactions ◽  
Design Method ◽  
Point Mutations ◽  
Computational Design ◽  
Data Bank ◽  
Protein Crystals ◽  
Single Individual ◽  
Space Group ◽  
Protein Backbones ◽  
Crystal Space

Substantial advances have been made in the computational design of protein interfaces over the last 20 years. However, the interfaces targeted by design have typically been stable and high-affinity. Here, we report the development of a generic computational design method to stabilize the weak interactions at crystallographic interfaces. Initially, we analyzed structures reported in the Protein Data Bank to determine whether crystals with more stable interfaces result in higher resolution structures. We found that for 22 variants of a single protein crystallized by a single individual, the Rosetta-calculated `crystal score' correlates with the reported diffraction resolution. We next developed and tested a computational design protocol, seeking to identify point mutations that would improve resolution in a highly stable variant of staphylococcal nuclease (SNase). Using a protocol based on fixed protein backbones, only one of the 11 initial designs crystallized, indicating modeling inaccuracies and forcing us to re-evaluate our strategy. To compensate for slight changes in the local backbone and side-chain environment, we subsequently designed on an ensemble of minimally perturbed protein backbones. Using this strategy, four of the seven designed proteins crystallized. By collecting diffraction data from multiple crystals per design and solving crystal structures, we found that the designed crystals improved the resolution modestly and in unpredictable ways, including altering the crystal space group. Post hoc, in silico analysis of the three observed space groups for SNase showed that the native space group was the lowest scoring for four of six variants (including the wild type), but that resolution did not correlate with crystal score, as it did in the preliminary results. Collectively, our results show that calculated crystal scores can correlate with reported resolution, but that the correlation is absent when the problem is inverted. This outcome suggests that more comprehensive modeling of the crystallographic state is necessary to design high-resolution protein crystals from poorly diffracting crystals.

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Miho Yuasa ◽  
Yosuke Nishikawa ◽  
Genji Kurisu ◽  
Shinobu Itoh ◽  
...  
Keyword(s):  
Michael Addition ◽  
In Silico ◽  
Addition Reaction ◽  
Single Point ◽  
Point Mutations ◽  
Unsaturated Ketone ◽  
Michael Addition Reaction ◽  
Beta Barrel ◽  
Cupin Superfamily ◽  
Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells

2021 ◽  
Author(s):  
Marisa L. Martino ◽  
Stephen N. Crooke ◽  
Marianne Manchester ◽  
M.G. Finn
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Virus Like Particles ◽  
Single Point Mutations

scholarly journals mCSM-membrane: predicting the effects of mutations on transmembrane proteins

2020 ◽  
Vol 48 (W1) ◽  
pp. W147-W153 ◽  
Author(s):  
Douglas E V Pires ◽  
Carlos H M Rodrigues ◽  
David B Ascher
Keyword(s):  
Membrane Proteins ◽  
Single Point ◽  
Point Mutations ◽  
Web Server ◽  
Protein Coding ◽  
Pathogenic Variants ◽  
Model Protein ◽  
Matthew’S Correlation Coefficient ◽  
Membrane Protein Stability ◽  
User Friendly

Abstract Significant efforts have been invested into understanding and predicting the molecular consequences of mutations in protein coding regions, however nearly all approaches have been developed using globular, soluble proteins. These methods have been shown to poorly translate to studying the effects of mutations in membrane proteins. To fill this gap, here we report, mCSM-membrane, a user-friendly web server that can be used to analyse the impacts of mutations on membrane protein stability and the likelihood of them being disease associated. mCSM-membrane derives from our well-established mutation modelling approach that uses graph-based signatures to model protein geometry and physicochemical properties for supervised learning. Our stability predictor achieved correlations of up to 0.72 and 0.67 (on cross validation and blind tests, respectively), while our pathogenicity predictor achieved a Matthew's Correlation Coefficient (MCC) of up to 0.77 and 0.73, outperforming previously described methods in both predicting changes in stability and in identifying pathogenic variants. mCSM-membrane will be an invaluable and dedicated resource for investigating the effects of single-point mutations on membrane proteins through a freely available, user friendly web server at http://biosig.unimelb.edu.au/mcsm_membrane.

scholarly journals In Vivo Evidence for TonB Dimerization

2003 ◽  
Vol 185 (19) ◽  
pp. 5747-5754 ◽  
Author(s):  
Annette Sauter ◽  
S. Peter Howard ◽  
Volkmar Braun
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Ferric Citrate ◽  
Site Directed Mutagenesis ◽  
Toxin Gene ◽  
Transmembrane Region ◽  
Dimer Formation ◽  
Citrate Transport ◽  
Hybrid Proteins

ABSTRACT TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and φ80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P ctx ::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P ctx ::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

2017 ◽  
Vol 474 (18) ◽  
pp. 3189-3205 ◽  
Author(s):  
Ashoka Chary Taviti ◽  
Tushar Kant Beuria
Keyword(s):  
Cell Division ◽  
Single Point ◽  
Point Mutations ◽  
Direct Interaction ◽  
Ring Formation ◽  
Z Ring ◽  
Single Point Mutations ◽  
Biochemical Analyses ◽  
Ftsz Assembly ◽  
The Mind

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

Frequency distribution of space groups in soluble and membrane proteins and their complexes

2021 ◽  
Vol 77 (6) ◽  
pp. 187-191
Author(s):  
Rajneesh K. Gaur
Keyword(s):  
Membrane Proteins ◽  
Frequency Distribution ◽  
Data Bank ◽  
Frequency Distributions ◽  
Space Group ◽  
Space Groups ◽  
Group Frequency ◽  
Different Types ◽  
Analytical Assessment ◽  
Three Space

The space-group frequency distributions for two types of proteins and their complexes are explored. Based on the incremental availability of data in the Protein Data Bank, an analytical assessment shows a preferential distribution of three space groups, i.e. P212121 > P1211 > C121, in soluble and membrane proteins as well as in their complexes. In membrane proteins, the order of the three space groups is P212121 > C121 > P1211. The distribution of these space groups also shows the same pattern whether a protein crystallizes with a monomer or an oligomer in the asymmetric unit. The results also indicate that the sizes of the two entities in the structures of soluble proteins crystallized as complexes do not influence the frequency distribution of space groups. In general, it can be concluded that the space-group frequency distribution is homogenous across different types of proteins and their complexes.

A Framework for Component Layout and Geometry Design of Mechanical Systems: Configuration Network and its Viewing Control

1995 ◽  
Author(s):  
Kikuo Fujita ◽  
Shinsuke Akagi
Keyword(s):  
Design Method ◽  
Mechanical Systems ◽  
Optimization Procedure ◽  
Computational Design ◽  
Object Oriented Programming ◽  
Design System ◽  
Air Conditioner ◽  
Programming Technique ◽  
Geometry Design ◽  
Component Layout

Abstract A Framework of computational design method and model is proposed for layout and geometry design of complicated mechanical systems, which is named “configuration network and its viewing control”. In the method, a design object is represented with a set of declarative relationships among various elements of a system, that is, configurations, which is gradually extended from schematic structure to exact layout and geometry through design process. Since a whole of such configurations forms a too complicated network to compute all together, how to view subparts is controlled based on levels of granularity and width of scope range. Such a configuration network is made to grow and refined through embodying geometry and layout corresponding to a focused subpart with a numerical optimization procedure. The framework has also an ability to flexibly integrate with engineering analysis. Moreover, a design system is implemented with an object-oriented programming technique, and it is applied to a design problem of air conditioner units in order to show the validity and effectiveness of the framework.

Computational Design for Digitally Fabricated 3D Inductive Power Transfer Coils

2022 ◽  
pp. 1-28
Author(s):  
Jun Xu ◽  
Eugeni L. Doubrovski ◽  
Jo Geraedts ◽  
Yu Song
Keyword(s):  
Design Method ◽  
Computational Design ◽  
Future Research ◽  
Receiver Coil ◽  
Power Transfer ◽  
Coil Design ◽  
Optimal Position ◽  
Inductive Power Transfer ◽  
Transmitter Coil ◽  
Inductive Power

Abstract The geometric shapes and the relative position of coils influence the performance of a three-dimensional (3D) inductive power transfer system. In this paper, we propose a coil design method for specifying the positions and the shapes of a pair of coils to transmit the desired power in 3D. Given region of interests (ROIs) for designing the transmitter and the receiver coils on two surfaces, the transmitter coil is generated around the center of its ROI first. The center of the receiver coil is estimated as a random seed position in the corresponding 3D surface. At this position, we use the heatmap method with electromagnetic constraints to iteratively extend the coil until the desired power can be transferred via the set of coils. In each step, the shape of the extension, i.e. a new turn of the receiver coil, is found as a spiral curve based on the convex hulls of adjacent turns in the 2D projection plane along their normal direction. Then, the optimal position of the receiver coil is found by maximizing the efficiency of the system. In the next step, the position and the shape of the transmitter coil are optimized based on the fixed receiver coil using the same method. This zig-zag optimization process iterates until an optimum is reached. Simulations and experiments with digitally fabricated prototypes were conducted and the effectiveness of the proposed 3D coil design method was verified. Possible future research directions are highlighted well.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

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