scholarly journals In Vivo Enzyme Entrapment in a Protein Crystal

2020 ◽  
Vol 142 (22) ◽  
pp. 9879-9883
Author(s):  
Bradley S. Heater ◽  
Zaofeng Yang ◽  
Marianne M. Lee ◽  
Michael K. Chan
Keyword(s):  
Protein Crystal ◽  
Enzyme Entrapment

Targeting DNA Repair Systems in Antitubercular Drug Development

2019 ◽  
Vol 26 (8) ◽  
pp. 1494-1505 ◽  
Author(s):  
Alina Minias ◽  
Anna Brzostek ◽  
Jarosław Dziadek
Keyword(s):  
Dna Repair ◽  
Protein Crystal ◽  
Model Organisms ◽  
Dna Repair Genes ◽  
Associated Proteins ◽  
Related Proteins ◽  
Repair Genes

Infections with Mycobacterium tuberculosis, the causative agent of tuberculosis, are difficult to treat using currently available chemotherapeutics. Clinicians agree on the urgent need for novel drugs to treat tuberculosis. In this mini review, we summarize data that prompts the consideration of DNA repair-associated proteins as targets for the development of new antitubercular compounds. We discuss data, including gene expression data, that highlight the importance of DNA repair genes during the pathogenic cycle as well as after exposure to antimicrobials currently in use. Specifically, we report experiments on determining the essentiality of DNA repair-related genes. We report the availability of protein crystal structures and summarize discovered protein inhibitors. Further, we describe phenotypes of available gene mutants of M. tuberculosis and model organisms Mycobacterium bovis and Mycobacterium smegmatis. We summarize experiments regarding the role of DNA repair-related proteins in pathogenesis and virulence performed both in vitro and in vivo during the infection of macrophages and animals. We detail the role of DNA repair genes in acquiring mutations, which influence the rate of drug resistance acquisition.

scholarly journals Molecular docking studies of some selected gallic acid derivatives against five non-structural proteins of novel coronavirus

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Haruna Isiyaku Umar ◽  
Bushra Siraj ◽  
Adeola Ajayi ◽  
Tajudeen O. Jimoh ◽  
Prosper Obed Chukwuemeka
Keyword(s):  
Binding Energy ◽  
Gallic Acid ◽  
High Plasma ◽  
Protein Crystal ◽  
World Health ◽  
Structural Proteins ◽  
Protein Database ◽  
Therapeutic Compounds

Abstract Background The World Health Organization has recently declared a new coronavirus disease (COVID-19) a pandemic and a global health emergency. The pressure to produce drugs and vaccines against the ongoing pandemic has resulted in the use of some drugs such as azithromycin, chloroquine (sulfate and phosphate), hydroxychloroquine, dexamethasone, favipiravir, remdesivir, ribavirin, ivermectin, and lopinavir/ritonavir. However, reports from some of the clinical trials with these drugs have proved detrimental on some COVID-19 infected patients with side effects more of which cardiomyopathy, cardiotoxicity, nephrotoxicity, macular retinopathy, and hepatotoxicity have been recently reported. Realizing the need for potent and harmless therapeutic compounds to combat COVID-19, we attempted in this study to find promising therapeutic compounds against the imminent threat of this virus. In this current study, 16 derivatives of gallic acid were docked against five selected non-structural proteins of SARS-COV-2 known to be a good target for finding small molecule inhibitors against the virus, namely, nsp3, nsp5, nsp12, nsp13, and nsp14. All the protein crystal structures and 3D structures of the small molecules (16 gallic acid derivatives and 3 control drugs) were retrieved from the Protein database (PDB) and PubChem server respectively. The compounds with lower binding energy than the control drugs were selected and subjected to pharmacokinetics screening using AdmetSAR server. Results 4-O-(6-galloylglucoside) gave binding energy values of − 8.4, − 6.8, − 8.9, − 9.1, and − 7.5 kcal/mol against Mpro, nsp3, nsp12, nsp13, and nsp15 respectively. Based on the ADMET profile, 4-O-(6-galloylglucoside) was found to be metabolized by the liver and has a very high plasma protein binding. Conclusion The result of this study revealed that 4-O-(6-galloylglucoside) could be a promising inhibitor against these SAR-Cov-2 proteins. However, there is still a need for further molecular dynamic simulation, in vivo and in vitro studies to support these findings.

scholarly journals In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130497 ◽  
Author(s):  
François-Xavier Gallat ◽  
Naohiro Matsugaki ◽  
Nathan P. Coussens ◽  
Koichiro J. Yagi ◽  
Marion Boudes ◽  
...  
Keyword(s):  
Structural Biology ◽  
Free Electron ◽  
Free Electron Laser ◽  
Protein Crystal ◽  
X Ray ◽  
Sample Characterization ◽  
The One ◽  
Serial Femtosecond Crystallography

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.

scholarly journals Combining X-rays, neutrons and electrons, and NMR, for precision and accuracy in structure–function studies

2021 ◽  
Vol 77 (3) ◽  
pp. 173-185
Author(s):  
John R. Helliwell
Keyword(s):  
Structure Function ◽  
Crystal Structures ◽  
Structure Prediction ◽  
Protein Crystal ◽  
X Rays ◽  
Time Resolved ◽  
X Ray ◽  
Precision And Accuracy ◽  
Electron Microscopes

The distinctive features of the physics-based probes used in understanding the structure of matter focusing on biological sciences, but not exclusively, are described in the modern context. This is set in a wider scope of holistic biology and the scepticism about `reductionism', what is called the `molecular level', and how to respond constructively. These topics will be set alongside the principles of accuracy and precision, and their boundaries. The combination of probes and their application together is the usual way of realizing accuracy. The distinction between precision and accuracy can be blurred by the predictive force of a precise structure, thereby lending confidence in its potential accuracy. These descriptions will be applied to the comparison of cryo and room-temperature protein crystal structures as well as the solid state of a crystal and the same molecules studied by small-angle X-ray scattering in solution and by electron microscopy on a sample grid. Examples will include: time-resolved X-ray Laue crystallography of an enzyme Michaelis complex formed directly in a crystal equivalent to in vivo; a new iodoplatin for radiation therapy predicted from studies of platin crystal structures; and the field of colouration of carotenoids, as an effective assay of function, i.e. their colouration, when unbound and bound to a protein. The complementarity of probes, as well as their combinatory use, is then at the foundation of real (biologically relevant), probe-artefacts-free, structure–function studies. The foundations of our methodologies are being transformed by colossal improvements in technologies of X-ray and neutron sources and their beamline instruments, as well as improved electron microscopes and NMR spectrometers. The success of protein structure prediction from gene sequence recently reported by CASP14 also opens new doors to change and extend the foundations of the structural sciences.

scholarly journals Structure of a heterogeneous, glycosylated, lipid-bound,in vivo-grown protein crystal at atomic resolution from the viviparous cockroachDiploptera punctata

IUCrJ ◽  
2016 ◽  
Vol 3 (4) ◽  
pp. 282-293 ◽  
Author(s):  
Sanchari Banerjee ◽  
Nathan P. Coussens ◽  
François-Xavier Gallat ◽  
Nitish Sathyanarayanan ◽  
Jandhyam Srikanth ◽  
...  
Keyword(s):  
Protein Structures ◽  
Milk Proteins ◽  
Living Organism ◽  
Atomic Resolution ◽  
Crystalline Lattice ◽  
Protein Crystal ◽  
X Ray ◽  
Recent Developments

Macromolecular crystals for X-ray diffraction studies are typically grownin vitrofrom pure and homogeneous samples; however, there are examples of protein crystals that have been identifiedin vivo. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grownin cellulo. Here, an atomic resolution (1.2 Å) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. Thesein vivo-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach,Diploptera punctata. The milk proteins crystallized in space groupP1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a singlein vivo-grown crystal of a natural protein in its native environment at atomic resolution.

scholarly journals Formation of a highly dense tetra-rhenium cluster in a protein crystal and its implications in medical imaging

IUCrJ ◽  
2019 ◽  
Vol 6 (4) ◽  
pp. 695-702 ◽  
Author(s):  
Alice Brink ◽  
John R. Helliwell
Keyword(s):  
Medical Imaging ◽  
Protein Crystal ◽  
Cluster Compound ◽  
Egg White Lysozyme ◽  
Starting Compound ◽  
Biological Compatibility ◽  
Rhenium Cluster ◽  
Rhenium Tricarbonyl

The fact that a protein crystal can serve as a chemical reaction vessel is intrinsically fascinating. That it can produce an electron-dense tetranuclear rhenium cluster compound from a rhenium tricarbonyl tribromo starting compound adds to the fascination. Such a cluster has been synthesized previously in vitro, where it formed under basic conditions. Therefore, its synthesis in a protein crystal grown at pH 4.5 is even more unexpected. The X-ray crystal structures presented here are for the protein hen egg-white lysozyme incubated with a rhenium tricarbonyl tribromo compound for periods of one and two years. These reveal a completed, very well resolved, tetra-rhenium cluster after two years and an intermediate state, where the carbonyl ligands to the rhenium cluster are not yet clearly resolved, after one year. A dense tetranuclear rhenium cluster, and its technetium form, offer enhanced contrast in medical imaging. Stimulated by these crystallography results, the unusual formation of such a species directly in an in vivo situation has been considered. It offers a new option for medical imaging compounds, particularly when considering the application of the pre-formed tetranuclear cluster, suggesting that it may be suitable for medical diagnosis because of its stability, preference of formation and biological compatibility.

scholarly journals Serial crystallography using synchrotron radiation - novel strategies for microcrystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C316-C316
Author(s):  
Cornelius Gati ◽  
Gleb Bourenkov ◽  
Marco Klinge ◽  
Dirk Rehders ◽  
Francesco Stellato ◽  
...  
Keyword(s):  
Structural Model ◽  
Structural Information ◽  
Protein Crystal ◽  
Array Detector ◽  
X Ray ◽  
Pixel Array ◽  
Serial Crystallography ◽  
Diffraction Patterns ◽  
Full Intensity

Protein crystallography continues to be one of the most frequently used techniques to obtain structural information of biomacromolecules to atomic resolution. Since protein crystals of delicate target systems are often limited in size, one of the main goals in the design of modern beamlines is the construction of highly intense X-ray beams with small focal size to obtain high resolution diffraction images of microcrystals. However, this development has led to the situation, that the full intensity of the beam can destroy a protein crystal within fractions of a second. Therefore often only a small number of diffraction patterns can be obtained from one single crystal. Here we describe the adaptation of the serial crystallography approach, which has first been developed at X-ray Free-Electron Lasers (Chapman et al. 2011) to the usage of a microfocus synchrotron beamline, using a standard cryogenic loop for sample delivery. We proved this concept with in vivo grown cathepsinB microcrystals (TbCatB, Koopmann et al. 2012, Redecke et al. 2013) (average of 9 μm3), a medically and pharmaceutically relevant protein, involved in the life cycle of T. brucei. In these experiments it was possible to show that serial crystallography enables the utilization and outcome of the above described bottlenecks and features of modern 3rd generation synchrotron microfocus beamlines. Our strategy exploits the combination of a micron-sized X-ray beam, high precision diffractometry and shutterless data acquisition with a pixel-array detector. By combining the data of 80 TbCatB crystals, it was possible to assemble a dataset to 3.0 Å resolution. The data allow the refinement of a structural model that is consistent with that previously obtained using FEL radiation, providing mutual validation.

scholarly journals Serial femtosecond X-ray diffraction of in vivo crystals in intact yeast cells

2014 ◽  
Vol 70 (a1) ◽  
pp. C570-C570
Author(s):  
Daniel Passon ◽  
Arjen Jakobi ◽  
Francesco Stellato ◽  
Mengning Liang ◽  
Kevin Knoops ◽  
...  
Keyword(s):  
Yeast Cells ◽  
Protein Crystal ◽  
Crystal Formation ◽  
Proof Of Concept ◽  
X Ray Diffraction ◽  
X Ray ◽  
In Situ Crystallization ◽  
Diffraction Patterns

Peroxisomes are membrane-enclosed organelles in eukaryotic cells with important roles in lipid metabolism and the scavenging of reactive oxygen species. Peroxisomes are capable of carrying an unusually high load of proteins, which under appropriate nutrient conditions results in the in situ crystallization of peroxisomal proteins in several yeast species and vertebrate hepatocytes [1,2]. In the methylotrophic yeast H.polymorpha, the predominant peroxisomal protein alcohol/methanol oxidase (AO) oligomerizes into octameric assemblies with a molecular mass of 600 kDa that spontaneously form 200-500 nm crystallites within peroxisomes [1]. We exposed H.polymorpha cell suspensions containing peroxisome-confined AO crystallites to femtosecond X-ray pulses at the Coherent X-ray Imaging (CXI) experimental endstation at the Linac Coherent Light Source. Peak detection routines mining the resulting scattering profiles identified >5000 Bragg-sampled diffraction patterns, providing the proof of concept that background scattering from the cells does not deteriorate the signal-to-noise ratio to an extent precluding observation of diffraction from individual AO crystallites. Summation patterns assembled from the individual frames match low-resolution powder diffraction patterns from concentrated suspensions of purified peroxisomes collected at the P14 beamline at the PETRAIII synchrotron, confirming that the observed diffraction mainly results from Bragg scattering of peroxisomal crystallites. To the best of our knowledge our data are the first to report room temperature X-ray diffraction from functional protein crystals in their native cellular environment. Currently the maximum resolution achieved in the diffraction patterns is limited to 20-15 Å. Future work will need to address improved sample preparation protocols in order to assess whether diffraction to a resolution sufficient to permit structure solution can be obtained. Protein crystal formation in vivo has been observed under physiological or pathological conditions in a number of other systems [3]. We hope that our results will help to establish serial femtosecond X-ray diffraction (SFX) as a method for structural characterization of cellular structures with crystalline content and provide a proof of concept for using in situ crystallization of proteins as a means to generate nanocrystalline samples for SFX.

Control of insects by bacteria

Parasitology ◽  
1982 ◽  
Vol 84 (4) ◽  
pp. 79-117 ◽  
Author(s):  
H. D. Burges
Keyword(s):  
Pest Control ◽  
Small Firms ◽  
Large Scale ◽  
Mosquito Control ◽  
Genetic Manipulation ◽  
Crop Damage ◽  
Protein Crystal ◽  
Stored Grain ◽  
Crystal Toxin

SUMMARYAll bacteria in microbial insecticides are species ofBacillusand form spores since they have to survive in the environment and on the shelf. They can be formulated as wettable powders, suspensions and dusts for application with conventional pest control machinery. All are safe to man and virtually all non-target organisms. Development costs are relatively low, but host specificity greatly restricts markets, the largest beingca. 2000 tons per annum in the West forB. thuringiensis. All act only after ingestion, a disadvantage because there is no contact action and usually only larvae are attacked. Three main groups have special features that determine their commercial success.TheB. popilliaegroup is produced onlyin vivowhich limits production by three small firms. The Japanese beetle has been controlled in grassland in the warm parts of the USA by single applications of spores in heaps, spaced 2 m each way. The bacterium spreads slowly to untreated areas, is very persistent and kills only by infection.TheB. thuringiensisgroup kills larvae of Lepidoptera, mosquitoes and blackflies, mainly by gut poisoning with a protein crystal toxin. It rapidly paralyses mouthparts and gut, stopping crop damage. It is readily produced by deep liquid fermentation, but does not persist and needs repeated application during the pest season. Products containing no beta exotoxin can be applied at unlimited dosage to food crops up to harvest. Only one application is needed for stored grain. After 20 years' use of strains against Lepidoptera, a different strain is now used commercially against mosquitoes and blackflies (only 5 years after its discovery), although improvements in formulation for aquatic application are needed. A recent new product based on the beta exotoxin is used in Finland only against flies in pig houses because it has some vertebrate toxicity.TheB. sphaericusgroup is similar toB. thuringiensis, except that its proteinaceous toxin is different, is situated in the spore wall in strain 1593, and attacks only mosquitoes. Now at the pilot production stage, its commercial future depends on whether it is more potent thanB. thuringiensisagainst certain species and whether it can recycle to give effective extended mosquito control in some environments.Intensive selection from natural isolates has improved potency 100 to 600 fold. This selective effort must be maintained and improved by genetic manipulation, which can be used to develop greater potential, particularly since DNA coding for the crystal toxin is carried on plasmids. This also gives speculative hope that the toxin may be incorporated into natural aquatic bacteria for mosquito control and into plants for protection against lepidopterous larvae. A great advantage is that these bacteria do not harm beneficial fauna to cause pest resurgence. At present, the main use lies in integrated pest control systems, although bacteria are not likely to supplant chemical insecticides on a large scale in the near future.

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