Real-Time Measurements of Protein Dynamics Using Fluorescence Activation-Coupled Protein Labeling Method

2011 ◽  
Vol 133 (17) ◽  
pp. 6745-6751 ◽  
Author(s):  
Toru Komatsu ◽  
Kai Johnsson ◽  
Hiroyuki Okuno ◽  
Haruhiko Bito ◽  
Takanari Inoue ◽  
...  
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Protein Labeling ◽  
Labeling Method

Real-time measurements of protein dynamics

2005 ◽  
Author(s):  
Caoimh��n G. Concannon ◽  
Heiko Duessmann ◽  
Jochen H. M. Prehn
Keyword(s):  
Real Time ◽  
Protein Dynamics

scholarly journals Physiological Study of Lactobacillus delbrueckii subsp. bulgaricus Strains in a Novel Chemically Defined Medium

2000 ◽  
Vol 66 (12) ◽  
pp. 5306-5311 ◽  
Author(s):  
Christian Chervaux ◽  
S. Dusko Ehrlich ◽  
Emmanuelle Maguin
Keyword(s):  
Carbon Sources ◽  
Streptococcus Thermophilus ◽  
Defined Medium ◽  
Lactobacillus Delbrueckii ◽  
Protein Labeling ◽  
Chemically Defined Medium ◽  
Phosphotransferase System ◽  
Nutritional Conditions ◽  
Labeling Method

ABSTRACT We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp.bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h−1. MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies ofL. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.

scholarly journals Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Shue Wang ◽  
Sagardip Majumder ◽  
Nicholas J Emery ◽  
Allen P Liu
Keyword(s):  
Synthetic Biology ◽  
Real Time ◽  
Protein Dynamics ◽  
Mammalian Cell ◽  
Locked Nucleic Acid ◽  
Free Expression ◽  
Reporter Protein ◽  
Bottom Up ◽  
Lna Probe

Abstract Transcription and translation are two critical processes during eukaryotic gene expression that regulate cellular activities. The development of mammalian cell-free expression (CFE) systems provides a platform for studying these two critical processes in vitro for bottom-up synthetic biology applications such as construction of an artificial cell. Moreover, real-time monitoring of the dynamics of synthesized mRNA and protein is key to characterize and optimize gene circuits before implementing in living cells or in artificial cells. However, there are few tools for measurement of mRNA and protein dynamics in mammalian CFE systems. Here, we developed a locked nucleic acid (LNA) probe for monitoring transcription in a HeLa-based CFE system in real-time. By using this LNA probe in conjunction with a fluorescent reporter protein, we were able to simultaneously monitor mRNA and protein dynamics in bulk reactions and cell-sized single-emulsion droplets. We found rapid production of mRNA transcripts that decreased over time as protein production ensued in bulk reactions. Our results also showed that transcription in cell-sized droplets has different dynamics compared to the transcription in bulk reactions. The use of this LNA probe in conjunction with fluorescent proteins in HeLa-based mammalian CFE system provides a versatile in vitro platform for studying mRNA dynamics for bottom-up synthetic biology applications.

scholarly journals Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Jennifer A. Martin ◽  
Yaroslav Chushak ◽  
Jorge L. Chávez ◽  
Joshua A. Hagen ◽  
Nancy Kelley-Loughnane
Keyword(s):  
Minimum Distance ◽  
Limit Of Detection ◽  
Signal To Noise Ratio ◽  
Protein Labeling ◽  
Target Interaction ◽  
Starting Conditions ◽  
Recognition Elements ◽  
Labeling Method ◽  
Biosensor Applications ◽  
Magnitude Improvement

Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface.

Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis

2010 ◽  
Vol 73 (10) ◽  
pp. 1986-2005 ◽  
Author(s):  
M. Fleron ◽  
Y. Greffe ◽  
D. Musmeci ◽  
A.C. Massart ◽  
V. Hennequiere ◽  
...  
Keyword(s):  
Protein Labeling ◽  
Labeling Method

scholarly journals Real Time de novo Deposition of Centromeric Histone-associated Proteins Using the Auxin Inducible Degradation system

2018 ◽  
Author(s):  
Sebastian Hoffmann ◽  
Daniele Fachinetti
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Protein Function ◽  
Genome Engineering ◽  
De Novo ◽  
Protein Complexes ◽  
Versatile Tool ◽  
Associated Proteins ◽  
Endogenous Proteins ◽  
Set Up

i.Summary/AbstractMeasuring protein dynamics is essential to uncover protein function and to understand the formation of large protein complexes such as centromeres. Recently, genome engineering in human cells has improved our ability to study the function of endogenous proteins. By combining genome editing techniques with the Auxin Inducible Degradation (AID) system, we created a versatile tool to study protein dynamics. This system allows us to analyze both protein function and dynamics by enabling rapid protein depletion and re-expression in the same experimental set-up. Here, we focus on the dynamics of the centromeric histone-associated protein CENP-C, responsible for the formation of the kinetochore complex. Following rapid removal and re-activation of a fluorescent version of CENP-C by auxin treatment and removal, we could follow CENP-C de novo deposition at centromeric regions during different stages of the cell cycle. In conclusion, the auxin degradation system is a powerful tool to assess and quantify protein dynamics in real time.

scholarly journals FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

2020 ◽  
Author(s):  
Matthieu Lagardère ◽  
Ingrid Chamma ◽  
Emmanuel Bouilhol ◽  
Macha Nikolski ◽  
Olivier Thoumine
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Simulated Data ◽  
Super Resolution ◽  
Molecular Complexes ◽  
Live Cell ◽  
Single Molecule Level ◽  
Resolution Imaging

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

scholarly journals AI-based Spectroscopic Monitoring of Real-time Interactions between SARS-CoV-2 and Human ACE2

2020 ◽  
Author(s):  
Sheng Ye ◽  
Guozhen Zhang ◽  
Jun Jiang
Keyword(s):  
Machine Learning ◽  
Real Time ◽  
Protein Dynamics ◽  
Structural Information ◽  
Protein Complexes ◽  
Spike Protein ◽  
Proof Of Concept ◽  
Spectroscopy Study ◽  
Time Resolved ◽  
Spectroscopic Monitoring

<div> <p>Here we demonstrate by a proof-of-concept simulation of IR spectra of complex of spike protein of SARS-CoV-2 and human ACE2, that a time-resolved spectroscopy may monitor the real-time structural information of the protein-protein complexes of interest, with the help of a machine learning protocol. The significant speedup of our approach relative to conventional quantum chemistry approach suggests a promising way of accelerating the development of real-time spectroscopy study of protein dynamics.</p> </div>

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