The NMR tube bioreactor

Chatzikonstantinou, Alexandra V.; Tsiailanis, Antonis D.; Gerothanassis, Ioannis P.; Stamatis, Haralambos; Ravera, Enrico; Fragai, Marco; Luchinat, Claudio; Parigi, Giacomo; Tzakos, Andreas G.

doi:10.1016/bs.mie.2019.10.032

Cited by 5 publications

(5 citation statements)

References 27 publications

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“…The in situ monitoring of the enzymatic reaction of the PCA or gallate biotransformation products was performed, for the first time, using a 5 mm NMR tube bioreactor [33,34]. For this, 1 mM of the substrate was added to 8 µM of PcaA in a 5 mm NMR tube and 1D and 2D NMR data were recorded.…”

Section: In Situ Monitoring Of the Pcaa Enzymatic Reaction Using Pca Or Gallate As Substrates In The Nmr Tube Bioreactormentioning

confidence: 99%

“…STD NMR methodology has previously been applied to simultaneously screen for interactions of the biotransformation products with protein targets in the NMR tube [33,34]. This NMR technique is based on the intermolecular transfer of magnetization from the irradiated protein receptor to the ligand resulting in the determination of interacting protons that are in proximity to the protein (≤5 Å) [35].…”

Section: In Situ Monitoring Of the Pcaa Enzymatic Reaction Using Pca Or Gallate As Substrates In The Nmr Tube Bioreactormentioning

confidence: 99%

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Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube

Tsagogiannis

Vandera

Primikyri

et al. 2021

IJMS

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The current study aims at the functional and kinetic characterization of protocatechuate (PCA) 4,5-dioxygenase (PcaA) from Pseudarthrobacter phenanthrenivorans Sphe3. This is the first single subunit Type II dioxygenase characterized in Actinobacteria. RT-PCR analysis demonstrated that pcaA and the adjacent putative genes implicated in the PCA meta-cleavage pathway comprise a single transcriptional unit. The recombinant PcaA is highly specific for PCA and exhibits Michaelis–Menten kinetics with Km and Vmax values of 21 ± 1.6 μM and 44.8 ± 4.0 U × mg−1, respectively, in pH 9.5 and at 20 °C. PcaA also converted gallate from a broad range of substrates tested. The enzymatic reaction products were identified and characterized, for the first time, through in situ biotransformation monitoring inside an NMR tube. The PCA reaction product demonstrated a keto-enol tautomerization, whereas the gallate reaction product was present only in the keto form. Moreover, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the pathway) were also determined, showing an induction when cells were grown on PCA and phenanthrene. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development.

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Section: In Situ Monitoring Of the Pcaa Enzymatic Reaction Using Pca Or Gallate As Substrates In The Nmr Tube Bioreactormentioning

confidence: 99%

Section: In Situ Monitoring Of the Pcaa Enzymatic Reaction Using Pca Or Gallate As Substrates In The Nmr Tube Bioreactormentioning

confidence: 99%

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube

Tsagogiannis

Vandera

Primikyri

et al. 2021

IJMS

Self Cite

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“…For example, the DNP method can enhance the sensitivity of NMR experiments by orders of magnitude and enable protein analysis at submicromolar concentrations . The NMR tube bioreactor can maintain cell viability for up to 24 h, avoid protein leakage during long-term in-cell experiments, and facilitate real time monitoring of biological processes . With further instrumental and methodological developments, we anticipate that in-cell NMR will become a powerful analytical tool to elucidate the structures and dynamics of proteins at physiological concentrations in native cellular environments and bridge the gap between in vitro and in vivo structural studies.…”

Section: Perspectivesmentioning

confidence: 99%

NMR-Based Methods for Protein Analysis

Cheng

et al. 2021

Anal. Chem.

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Nuclear magnetic resonance (NMR) spectroscopy is a well-established method for analyzing protein structure, interaction, and dynamics at atomic resolution and in various sample states including solution state, solid state, and membranous environment. Thanks to rapid NMR methodology development, the past decade has witnessed a growing number of protein NMR studies in complex systems ranging from membrane mimetics to living cells, which pushes the research frontier further toward physiological environments and offers unique insights in elucidating protein functional mechanisms. In particular, in-cell NMR has become a method of choice for bridging the huge gap between structural biology and cell biology. Herein, we review the recent developments and applications of NMR methods for protein analysis in close-to-physiological environments, with special emphasis on in-cell protein structural determination and the analysis of protein dynamics, both difficult to be accessed by traditional methods.

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“…This can be challenging for a novice operator due to the stabilization requirements. The system is based on an open-source package [12] developed using the PyBullet physics engine. The observation space is a three-dimensional continuous vector that includes the angular position and velocity of the robot's body, and the linear velocity of the system.…”

Section: Experimental Evaluationmentioning

confidence: 99%

Highly Parallelized Data-Driven MPC for Minimal Intervention Shared Control

Broad¹,

Murphey²,

Argall³

2019

Robotics: Science and Systems XV

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We present a shared control paradigm that improves a user's ability to operate complex, dynamic systems in potentially dangerous environments without a priori knowledge of the user's objective. In this paradigm, the role of the autonomous partner is to improve the general safety of the system without constraining the user's ability to achieve unspecified behaviors. Our approach relies on a data-driven, model-based representation of the joint human-machine system to evaluate, in parallel, a significant number of potential inputs that the user may wish to provide. These samples are used to (1) predict the safety of the system over a receding horizon, and (2) minimize the influence of the autonomous partner. The resulting shared control algorithm maximizes the authority allocated to the human partner to improve their sense of agency, while improving safety. We evaluate the efficacy of our shared control algorithm with a human subjects study (n=20) conducted in two simulated environments: a balance bot and a race car. During the experiment, users are free to operate each system however they would like (i.e., there is no specified task) and are only asked to try to avoid unsafe regions of the state space. Using modern computational resources (i.e., GPUs) our approach is able to consider more than 10,000 potential trajectories at each time step in a control loop running at 100Hz for the balance bot and 60Hz for the race car. The results of the study show that our shared control paradigm improves system safety without knowledge of the user's goal, while maintaining high-levels of user satisfaction and low-levels of frustration. Our code is available online at https://github.com/asbroad/mpmi shared control.

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The NMR tube bioreactor

Cited by 5 publications

References 27 publications

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube

NMR-Based Methods for Protein Analysis

Highly Parallelized Data-Driven MPC for Minimal Intervention Shared Control

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