scholarly journals Chemical clustering and visualization applied to macromolecular crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1145-C1145
Author(s):  
Andrew Bruno ◽  
Amanda Ruby ◽  
Joseph Luft ◽  
Thomas Grant ◽  
Jayaraman Seetharaman ◽  
...  
Keyword(s):  
Hierarchical Clustering ◽  
High Throughput ◽  
Active Sites ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Potential Applications ◽  
Crystallization Conditions ◽  
Electron Density Map ◽  
Distance Coefficient ◽  
Rate Limiting

Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and the insertion and correct placement of a sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient appears to be robust in this application and coupled with hierarchical clustering and the overlay of crystallization outcome reveals information of biological relevance. While tested with a single example the potential applications appear promising.

scholarly journals A beginner’s guide to macromolecular crystallization

2021 ◽  
Vol 43 (1) ◽  
pp. 36-43
Author(s):  
Fabrice Gorrec
Keyword(s):  
Crystal Nucleation ◽  
Trial And Error ◽  
Bioinformatics Analyses ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Conditions ◽  
Macromolecular Crystallization ◽  
Rate Limiting

Obtaining diffraction-quality crystals is currently the rate-limiting step in macromolecular X-ray crystallography of proteins, DNA, RNA or their complexes, in the vast majority of cases. Since each sample has different and specific characteristics – which is the reason for wanting to study every single one of them in the first place – crystallization conditions cannot be predicted. Hence, researchers must enable crystal nucleation and growth through experimentation and screening. The size, shape and surface of the sample or complexes of interest are often altered through genetic and biochemical manipulation to facilitate crystallization, based on bioinformatics analyses and trial and error. Pure samples are trialled against a very broad range of crystallization conditions. The currently predominant method to achieve crystallization is sitting drop vapour diffusion with nanolitre-class robotic liquid handlers. Once initial screening yields crystals, further optimization experiments are usually required to obtain larger and diffraction-quality crystals.

scholarly journals Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion inSLC6A3

2019 ◽  
Vol 116 (9) ◽  
pp. 3853-3862 ◽  
Author(s):  
Nicholas G. Campbell ◽  
Aparna Shekar ◽  
Jenny I. Aguilar ◽  
Dungeng Peng ◽  
Vikas Navratna ◽  
...  
Keyword(s):  
Autism Spectrum ◽  
High Time Resolution ◽  
Paramagnetic Resonance ◽  
X Ray Crystallography ◽  
Fine Grained ◽  
Resonance Analysis ◽  
Rate Limiting Step ◽  
Abnormal Behaviors ◽  
Rate Limiting ◽  
Electron Paramagnetic

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a “half-open and inward-facing” state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains ofDrosophila melanogasterexpressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

scholarly journals Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway

2006 ◽  
Vol 72 (3) ◽  
pp. 1949-1955 ◽  
Author(s):  
Jorge Carpinelli ◽  
Reinhard Krämer ◽  
Eduardo Agosin
Keyword(s):  
Corynebacterium Glutamicum ◽  
Specific Productivity ◽  
Partial Recovery ◽  
Glycogen Accumulation ◽  
Production Methods ◽  
Homologous Overexpression ◽  
Rate Limiting Step ◽  
Potential Applications ◽  
Rate Limiting

ABSTRACT Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.

Crystallization of protein–protein complexes

2002 ◽  
Vol 35 (6) ◽  
pp. 674-676 ◽  
Author(s):  
Sergei Radaev ◽  
Peter D. Sun
Keyword(s):  
Sparse Matrix ◽  
Protein Complexes ◽  
Data Bank ◽  
Structure Characterization ◽  
Salt Crystallization ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Conditions ◽  
The Stability ◽  
Rate Limiting

Crystallizing protein–protein complexes remains a rate-limiting step in their structure characterization. Crystallization conditions for the known protein–protein complexes have been surveyed in both the Protein Data Bank and the BMCD database. Compared with non-complexed proteins, crystallization conditions for protein–protein complexes are less diverse and heavily favor (71%versus27%) polyethylene glycols (PEG) rather than ammonium sulfate or other high-salt crystallization conditions. The results suggest that the stability of protein complexes limits their available crystallization configuration space. Based on the survey, a set of sparse-matrix screen conditions was designed.

scholarly journals Co-operativity in seminal ribonuclease function: binding studies

1987 ◽  
Vol 241 (2) ◽  
pp. 435-440 ◽  
Author(s):  
A Di Donato ◽  
R Piccoli ◽  
G D'Alessio
Keyword(s):  
Active Sites ◽  
Mixed Type ◽  
Equilibrium Dialysis ◽  
Binding Studies ◽  
Rate Limiting Step ◽  
Differential Spectrophotometry ◽  
Limiting Step ◽  
Binding Data ◽  
Hysteretic Effect ◽  
Rate Limiting

Binding of nucleotides to bovine seminal RNAase was studied by differential spectrophotometry and equilibrium dialysis. Cytidine 3′-phosphate, the reaction product of the hydrolytic, rate-limiting step of the reaction, was found to be capable, in contrast to related nucleotides, of discriminating between the two structurally identical active sites of the enzyme. Negative co-operativity, with a ‘half-of-sites’ reactivity, was found at lower concentrations of ligand, whereas at higher concentrations positive co-operativity was detected. These findings exclude that the non-hyperbolic kinetics previously reported for the hydrolytic step of the reaction are due to hysteretic effect. A model of mixed-type co-operativity is proposed for interpreting the binding data.

scholarly journals Towards Novel Herbicide Modes of Action by Inhibiting Lysine Biosynthesis in Plants

2021 ◽  
Author(s):  
Tatiana P. Soares da Costa ◽  
Cody J. Hall ◽  
Santosh Panjikar ◽  
Jessica A. Wyllie ◽  
Rebecca M. Christoff ◽  
...  
Keyword(s):  
Herbicidal Activity ◽  
Lysine Biosynthesis ◽  
Proof Of Concept ◽  
Herbicide Resistant ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Promising Target ◽  
Chemical Screen ◽  
Germination And Growth ◽  
Rate Limiting

Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. Using a high throughput chemical screen, we identified the first class of plant DHDPS inhibitors that have micromolar potency against Arabidopsis thaliana DHDPS isoforms. Employing X-ray crystallography, we determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. We also demonstrated that the inhibitors attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide resistant weeds.

scholarly journals Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

2021 ◽  
Author(s):  
Mrinal Shekhar ◽  
Chitrak Gupta ◽  
Kano Suzuki ◽  
Abhishek Singharoy ◽  
Takeshi Murata
Keyword(s):  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Hydrolysis Product ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Dynamics Simulations ◽  
Rate Limiting

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or 'dwell' is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transfor-mations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain level interactions transcend into larger scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

Improving photosynthesis through the enhancement of Rubisco carboxylation capacity

2021 ◽  
Author(s):  
Concepción Iñiguez ◽  
Pere Aguiló-Nicolau ◽  
Jeroni Galmés
Keyword(s):  
Active Sites ◽  
Arable Land ◽  
Small Subunit ◽  
Resource Use Efficiency ◽  
Rate Limiting Step ◽  
Promising Target ◽  
Use Efficiency ◽  
Co2 Concentrating Mechanisms ◽  
Near Future ◽  
Rate Limiting

Rising human population, along with the reduction in arable land and the impacts of global change, sets out the need for continuously improving agricultural resource use efficiency and crop yield (CY). Bioengineering approaches for photosynthesis optimization have largely demonstrated the potential for enhancing CY. This review is focused on the improvement of Rubisco functioning, which catalyzes the rate-limiting step of CO2 fixation required for plant growth, but also catalyzes the ribulose-bisphosphate oxygenation initiating the carbon and energy wasteful photorespiration pathway. Rubisco carboxylation capacity can be enhanced by engineering the Rubisco large and/or small subunit genes to improve its catalytic traits, or by engineering the mechanisms that provide enhanced Rubisco expression, activation and/or elevated [CO2] around the active sites to favor carboxylation over oxygenation. Recent advances have been made in the expression, assembly and activation of foreign (either natural or mutant) faster and/or more CO2-specific Rubisco versions. Some components of CO2 concentrating mechanisms (CCMs) from bacteria, algae and C4 plants has been successfully expressed in tobacco and rice. Still, none of the transformed plant lines expressing foreign Rubisco versions and/or simplified CCM components were able to grow faster than wild type plants under present atmospheric [CO2] and optimum conditions. However, the results obtained up to date suggest that it might be achievable in the near future. In addition, photosynthetic and yield improvements have already been observed when manipulating Rubisco quantity and activation degree in crops. Therefore, engineering Rubisco carboxylation capacity continues being a promising target for the improvement in photosynthesis and yield.

scholarly journals Validation of Flory-Huggins model for phenol adsorption by Parthenium hysterophorus in a batch system

2019 ◽  
Vol 2 (2) ◽  
Author(s):  
Zakia Latif1 ◽  
Aliya Fazal2 ◽  
Muhammad Aziz Choudhary1 ◽  
Zahoor Ahmad1 ◽  
Muhammad Aslam Mirza1
Keyword(s):  
Active Sites ◽  
Cost Effective ◽  
Toxic Waste ◽  
Parthenium Hysterophorus ◽  
Phenol Adsorption ◽  
Rate Limiting Step ◽  
Adsorbent Surface ◽  
Ft Ir ◽  
Biomass Dosage ◽  
Rate Limiting

Parthenium hysterophorus weed powder was studied as adsorbent for phenol adsorption from its aqueous standardized solution. The adsorption of pollutant was found improving with an increase of biomass dosage and contact time. The intraparticle diffusion of phenol onto adsorbent surface was identified to be the rate limiting step. Linear form of Flory-Huggins model revealed preeminence to Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich due to highest value of R2. The remediation process was figured out as a physisorption rather than a chemical one based on value of E (0.21KJ/mol). Active sites of sorbent surface identified by FT-IR were oxygen containing functional groups. Recent study proposes cost effective utilization of toxic allergent for treatment of toxic waste.

scholarly journals Effect of the Preparation Method of LaSrCoFeOx Perovskites on the Activity of N2O Decomposition

2021 ◽  
Author(s):  
Nia Richards ◽  
Luke A. Parker ◽  
James H. Carter ◽  
Samuel Pattisson ◽  
David J. Morgan ◽  
...  
Keyword(s):  
Citric Acid ◽  
Active Sites ◽  
Preparation Method ◽  
N2o Decomposition ◽  
Oxygen Species ◽  
Calcination Temperatures ◽  
Highly Active ◽  
Rate Limiting Step ◽  
Enhanced Mobility ◽  
Rate Limiting

AbstractN2O remains a major greenhouse gas and contributor to global warming, therefore developing a catalyst that can decompose N2O at low temperatures is of global importance. We have investigated the use of LaSrCoFeOx perovskites for N2O decomposition and the effect of surface area, A and B site elements, Co–O bond strength, redox capabilities and oxygen mobility have been studied. It was found that by using a citric acid preparation method, perovskites with strong redox capabilities and weak Co–O bonds can be formed at relatively low calcination temperatures (550 °C) resulting in highly active catalysts. The enhanced activity is related to the presence of highly mobile oxygen species. Oxygen recombination on the catalyst surface is understood to be a prominent rate limiting step for N2O decomposition. Here the reduced strength of Co–O bonds and mobile lattice oxygen species suggest that the surface oxygen species have enhanced mobility, aiding recombination, and subsequent regeneration of the active sites. La0.75Sr0.25Co0.81Fe0.19Ox prepared by citric acid method converted 50% of the N2O in the feed (T50) at 448 °C. Graphic Abstract

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