scholarly journals Effect of Type I Antifreeze Proteins on the Freezing and Melting Processes of Cryoprotective Solutions Studied by Site-Directed Spin Labeling Technique

Crystals ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 352 ◽  
Author(s):  
Adiel F. Perez ◽  
Kyle R. Taing ◽  
Justin C. Quon ◽  
Antonia Flores ◽  
Yong Ba
Keyword(s):  
Freezing Point ◽  
Antifreeze Proteins ◽  
Active Role ◽  
Spin Labeling ◽  
Ice Crystals ◽  
Freezing Injury ◽  
Type I ◽  
Paramagnetic Resonance ◽  
Potential Applications ◽  
Site Directed Spin Labeling

Antifreeze proteins (AFPs) protect organisms living in subzero environments from freezing injury, which render them potential applications for cryopreservation of living cells, organs, and tissues. Cryoprotective agents (CPAs), such as glycerol and propylene glycol, have been used as ingredients to treat cellular tissues and organs to prevent ice crystal’s formation at low temperatures. To assess AFP’s function in CPA solutions, we have the applied site-directed spin labeling technique to a Type I AFP. A two-step process to prevent bulk freezing of the CPA solutions was observed by the cryo-photo microscopy, i.e., (1) thermodynamic freezing point depression by the CPAs; and (2) inhibition to the growth of seed ice crystals by the AFP. Electron paramagnetic resonance (EPR) experiments were also carried out from room temperature to 97 K, and vice versa. The EPR results indicate that the spin labeled AFP bound to ice surfaces, and inhibit the growths of ice through the bulk freezing processes in the CPA solutions. The ice-surface bound AFP in the frozen matrices could also prevent the formation of large ice crystals during the melting processes of the solutions. Our study illustrates that AFPs can play an active role in CPA solutions for cryopreservation applications.

scholarly journals Antifreeze Proteins and Their Practical Utilization in Industry, Medicine, and Agriculture

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1649
Author(s):  
Azadeh Eskandari ◽  
Thean Chor Leow ◽  
Mohd Basyaruddin Abdul Rahman ◽  
Siti Nurbaya Oslan
Keyword(s):  
Freezing Point ◽  
Proper Time ◽  
Antifreeze Proteins ◽  
Ice Crystals ◽  
Practical Utilization ◽  
Ice Recrystallization ◽  
Potential Applications ◽  
Specific Proteins ◽  
Β Sheets ◽  
Different Sources

Antifreeze proteins (AFPs) are specific proteins, glycopeptides, and peptides made by different organisms to allow cells to survive in sub-zero conditions. AFPs function by reducing the water’s freezing point and avoiding ice crystals’ growth in the frozen stage. Their capability in modifying ice growth leads to the stabilization of ice crystals within a given temperature range and the inhibition of ice recrystallization that decreases the drip loss during thawing. This review presents the potential applications of AFPs from different sources and types. AFPs can be found in diverse sources such as fish, yeast, plants, bacteria, and insects. Various sources reveal different α-helices and β-sheets structures. Recently, analysis of AFPs has been conducted through bioinformatics tools to analyze their functions within proper time. AFPs can be used widely in various aspects of application and have significant industrial functions, encompassing the enhancement of foods’ freezing and liquefying properties, protection of frost plants, enhancement of ice cream’s texture, cryosurgery, and cryopreservation of cells and tissues. In conclusion, these applications and physical properties of AFPs can be further explored to meet other industrial players. Designing the peptide-based AFP can also be done to subsequently improve its function.

scholarly journals Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions

2020 ◽  
Vol 11 (35) ◽  
pp. 9655-9664
Author(s):  
Yan Wang ◽  
Venkatesan Kathiresan ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Epr Spectroscopy ◽  
Base Pair ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic

Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date.

scholarly journals Characterization of the Linker 2 Region in Human Vimentin Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance†

Biochemistry ◽  
2006 ◽  
Vol 45 (39) ◽  
pp. 11737-11743 ◽  
Author(s):  
John F. Hess ◽  
Madhu S. Budamagunta ◽  
Rebecca L. Shipman ◽  
Paul G. FitzGerald ◽  
John C. Voss
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic

scholarly journals A fully enzymatic method for site-directed spin labeling of long RNA

2014 ◽  
Vol 42 (15) ◽  
pp. e117-e117 ◽  
Author(s):  
Isabelle Lebars ◽  
Bertrand Vileno ◽  
Sarah Bourbigot ◽  
Philippe Turek ◽  
Philippe Wolff ◽  
...  
Keyword(s):  
Coupling Reaction ◽  
Spin Labeling ◽  
In Vitro Transcription ◽  
Enzymatic Method ◽  
Paramagnetic Resonance ◽  
T4 Rna Ligase ◽  
Labeled Rna ◽  
Site Directed Spin Labeling ◽  
Novel Strategy

Abstract Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5′-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies.

scholarly journals Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

2019 ◽  
Author(s):  
Pia Widder ◽  
Julian Schuck ◽  
Daniel Summerer ◽  
Malte Drescher
Keyword(s):  
Spin Labeling ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Determination ◽  
Bioorthogonal Labeling ◽  
In Vitro Measurements ◽  
Site Directed Spin Labeling ◽  
Double Electron Electron Resonance

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E.coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.<br>

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