scholarly journals Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases

Crystals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 975
Author(s):  
Brigitte Walla ◽  
Daniel Bischoff ◽  
Robert Janowski ◽  
Nikolas von den Eichen ◽  
Dierk Niessing ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Crystal Packing ◽  
Protein Crystallization ◽  
Lactobacillus Brevis ◽  
Protein Crystal ◽  
Wild Type ◽  
Alcohol Dehydrogenases ◽  
Crystallization Conditions ◽  
Contact Engineering ◽  
Crystal Contact

Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of Lactobacillus brevis (LbADH) to Lactobacillus kefir (LkADH). Proteins were expressed in E. coli and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of LkADH wild type compared to LbADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of LkADH mutants with the wild type, the mean protein crystal size of LkADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the LkADH variants studied results in an order of crystallizability T102E > Q126K > LkADH wild type, which corresponds to the results with LbADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies.

Rational Crystal Contact Engineering ofLactobacillus brevisAlcohol Dehydrogenase To Promote Technical Protein Crystallization

2019 ◽  
Vol 19 (4) ◽  
pp. 2380-2387 ◽  
Author(s):  
Phillip Nowotny ◽  
Johannes Hermann ◽  
Jianing Li ◽  
Angela Krautenbacher ◽  
Kai Klöpfer ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
Contact Engineering ◽  
Crystal Contact

scholarly journals Automating the application of smart materials for protein crystallization

2015 ◽  
Vol 71 (3) ◽  
pp. 534-540 ◽  
Author(s):  
Sahir Khurshid ◽  
Lata Govada ◽  
Hazim F. EL-Sharif ◽  
Subrayal M. Reddy ◽  
Naomi E. Chayen
Keyword(s):  
Smart Materials ◽  
Molecularly Imprinted Polymers ◽  
Protein Crystallization ◽  
Nucleating Agent ◽  
Protein Crystal ◽  
Molecularly Imprinted ◽  
Imprinted Polymers ◽  
Protein Crystal Growth ◽  
Probability Of Success ◽  
Crystallization Conditions

The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as `smart materials') for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials.

Effective Improvement of D-Phenylglycine Aminotransferase Solubility by Protein Crystal Contact Engineering

2012 ◽  
Vol 22 (3) ◽  
pp. 147-155 ◽  
Author(s):  
Aiya Chantarasiri ◽  
Vithaya Meevootisom ◽  
Duangnate Isarangkul ◽  
Suthep Wiyakrutta
Keyword(s):  
Protein Crystal ◽  
Contact Engineering ◽  
Crystal Contact

scholarly journals Controlling Protein Crystallization by Free Energy Guided Design of Interactions at Crystal Contacts

Crystals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 588
Author(s):  
Johannes Hermann ◽  
Daniel Bischoff ◽  
Phillip Grob ◽  
Robert Janowski ◽  
Dariusch Hekmat ◽  
...  
Keyword(s):  
Free Energy ◽  
Protein Interactions ◽  
Computational Study ◽  
Protein Crystallization ◽  
Lactobacillus Brevis ◽  
Protein Crystal ◽  
Crystal Formation ◽  
Contact Stability ◽  
Crystal Contacts ◽  
Energy Simulations

Protein crystallization can function as an effective method for protein purification or formulation. Such an application requires a comprehensive understanding of the intermolecular protein–protein interactions that drive and stabilize protein crystal formation to ensure a reproducible process. Using alcohol dehydrogenase from Lactobacillus brevis (LbADH) as a model system, we probed in our combined experimental and computational study the effect of residue substitutions at the protein crystal contacts on the crystallizability and the contact stability. Increased or decreased contact stability was calculated using molecular dynamics (MD) free energy simulations and showed excellent qualitative correlation with experimentally determined increased or decreased crystallizability. The MD simulations allowed us to trace back the changes to their physical origins at the atomic level. Engineered charge–charge interactions as well as engineered hydrophobic effects could be characterized and were found to improve crystallizability. For example, the simulations revealed a redesigning of a water mediated electrostatic interaction (“wet contact”) into a water depleted hydrophobic effect (“dry contact”) and the optimization of a weak hydrogen bonding contact towards a strong one. These findings explained the experimentally found improved crystallizability. Our study emphasizes that it is difficult to derive simple rules for engineering crystallizability but that free energy simulations could be a very useful tool for understanding the contribution of crystal contacts for stability and furthermore could help guide protein engineering strategies to enhance crystallization for technical purposes.

scholarly journals Berkeley Screen: a set of 96 solutions for general macromolecular crystallization

2017 ◽  
Vol 50 (5) ◽  
pp. 1352-1358 ◽  
Author(s):  
Jose H. Pereira ◽  
Ryan P. McAndrew ◽  
Giovani P. Tomaleri ◽  
Paul D. Adams
Keyword(s):  
Crystal Packing ◽  
Protein Structures ◽  
Data Bank ◽  
Protein Crystal ◽  
Crystal Formation ◽  
Density Maps ◽  
Crystallization Experiments ◽  
Particular Solution ◽  
Crystallization Conditions ◽  
Macromolecular Crystallization

Using statistical analysis of the Biological Macromolecular Crystallization Database, combined with previous knowledge about crystallization reagents, a crystallization screen called the Berkeley Screen has been created. Correlating crystallization conditions and high-resolution protein structures, it is possible to better understand the influence that a particular solution has on protein crystal formation. Ions and small molecules such as buffers and precipitants used in crystallization experiments were identified in electron density maps, highlighting the role of these chemicals in protein crystal packing. The Berkeley Screen has been extensively used to crystallize target proteins from the Joint BioEnergy Institute and the Collaborative Crystallography program at the Berkeley Center for Structural Biology, contributing to several Protein Data Bank entries and related publications. The Berkeley Screen provides the crystallographic community with an efficient set of solutions for general macromolecular crystallization trials, offering a valuable alternative to the existing commercially available screens.

Screening and optimization of protein crystallization conditions through gradual evaporation using a novel crystallization platform

2005 ◽  
Vol 38 (6) ◽  
pp. 988-995 ◽  
Author(s):  
Sameer Talreja ◽  
David Y. Kim ◽  
Amir Y. Mirarefi ◽  
Charles F. Zukoski ◽  
Paul J. A. Kenis
Keyword(s):  
Phase Transition ◽  
High Throughput Screening ◽  
Large Fraction ◽  
Protein Crystallization ◽  
Rapid Screening ◽  
Successful Implementation ◽  
X Ray Diffraction ◽  
X Ray ◽  
Wide Range ◽  
Crystallization Conditions

High-throughput screening of a wide range of different conditions is typically required to obtain X-ray quality crystals of proteins for structure–function studies. The outcomes of individual experiments,i.e.the formation of gels, precipitates, microcrystals, or crystals, guide the search for and optimization of conditions resulting in X-ray diffraction quality crystals. Unfortunately, the protein will remain soluble in a large fraction of the experiments. In this paper, an evaporation-based crystallization platform is reported in which droplets containing protein and precipitant are gradually concentrated through evaporation of solvent until the solvent is completely evaporated. A phase transition is thus ensured for each individual crystallization compartment; hence the number of experiments and the amount of precious protein needed to identify suitable crystallization conditions is reduced. The evaporation-based method also allows for rapid screening of different rates of supersaturation, a parameter known to be important for optimization of crystal growth and quality. The successful implementation of this evaporation-based crystallization platform for identification and especially optimization of crystallization conditions is demonstrated using the model proteins of lysozyme and thaumatin.

scholarly journals Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 576
Author(s):  
Sofia Giacosa ◽  
Catherine Pillet ◽  
Irinka Séraudie ◽  
Laurent Guyon ◽  
Yann Wallez ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Renal Carcinoma ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Cancer Therapeutics ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Cell Renal Cell Carcinoma ◽  
Von Hippel Lindau ◽  
Renal Carcinoma Cells

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL− cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug–gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

Purification and characterization of a nonpolymerizing long-pitch actin dimer

2006 ◽  
Vol 84 (5) ◽  
pp. 695-702 ◽  
Author(s):  
Braden Sweeting ◽  
John F. Dawson
Keyword(s):  
Critical Concentration ◽  
Dnase I ◽  
Actin Binding ◽  
Wild Type ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Fold Reduction ◽  
Crystallization Conditions ◽  
Self Association ◽  
Pointed End

Atomic resolution structures of filamentous actin have not been obtained owing to the self-association of actin under crystallization conditions. Obtaining short filamentous actin complexes of defined lengths is therefore a highly desirable goal. Here we report the production and isolation of a long-pitch actin dimer employing chemical crosslinking between wild-type actin and Q41C/C374A mutant actin. The Q41C/C374A mutant actin possessed altered polymerization properties, with a 2-fold reduction in the rate of elongation and an increased critical concentration relative to wild-type actin. The Q41C/C374A mutant actin also displayed an increase in the IC50 for DNase I, a pointed-end actin-binding protein. The long-pitch dimer was bound by DNase I to prevent polymerization and purified. It was found that each actin dimer is bound by 2 DNase I molecules, 1 likely bound to each of the actin protomers. The long-pitch dimer bound by DNase I did not form short F actin structures, as assessed by the binding of rhodamine–phalloidin.

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