eoe sub articles

EoE Sub Articles

1. Production of 15N, 13C-Tau (Figure 1)
<ol>
	<li>ATransform pET15b-Tau recombinant T7 expression plasmid into BL21(DE3) competent Escherichia coli bacterial cells. 
	NOTE: the cDNA coding for the longest (441 amino acid residues) Tau isoform is cloned between NcoI and XhoI restriction sites in the pET15b plasmid.
	<ol>
		<li>Mix gently 50 &#181;l of competent BL21(DE3) cells, forming 1-5 x 107 colonies per &#181;g of pl...

nuclear magnetic resonance spectroscopy to identify multiple phosphorylations in proteins

Source: Danis, C. et. al., <a href="https://app.jove.com/v/55001">Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins.</a>&#160;J. Vis. Exp.&#160; (2016).
This video demonstrates the use of nuclear magnetic resonance spectroscopy (NMR) techniques to identify multiple phosphorylations in a protein. The phosphorylation of a protein at specific amino acid causes the deshielding of the neighboring amide hydrogen, which generates the spectral difference.

<img alt="Figure 1" src="/files/ftp_upload/21467/21467_Figure1.jpg" /> 
Figure 1: Scheme of the main steps of recombinant protein production and isotopic labeling.&#160;Steps from bacteria transformation to recombinant protein production are outlined as described in paragraph 1 of the protocol.
<img alt="Figure 2" src="/files/ftp_upload/21467/21467_Figure2.jpg" /> 
Figure 2: Scheme of the main ...

Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins

Source: Danis, C. et. al., Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered ...

Multiple phosphorylations in a protein involve adding phosphate groups to protein at various positions, specifically at serine and threonine residues.
To identify multiple phosphorylations in a protein, begin with a tube containing phosphorylated proteins labeled with nitrogen-15, which helps to differentiate amide hydrogens from other hydrogens in the protein.
Add an internal NMR reference compound for accurate measurement of the relative resonance frequencies. Transfer the mixture to a nuclear magnetic resonance, or NMR, tube. Insert the tube into a spinning aperture, and place it in an NMR spectrometer.
The presence of phosphates exhibits electron-withdrawing effects in the phosphorylated serine and threonine residues. As a result, the amide hydrogens of phosphorylated residues appear to be deshielded with lesser electron densities compared to the other amide hydrogens.
Amide hydrogens with varying electronic environments experience differential magnetic fields around nuclei, attaining low-energy states.
Apply a brief radiofrequency pulse to excite the hydrogen nuclei to a higher energy state at a specific resonance frequency based on their electronic environment.
During relaxation, the nuclei return to a low-energy state by releasing the absorbed energy, which is detected to produce an NMR signal.
Plot a two-dimensional NMR spectrum representing the individual amide hydrogens.
Compare the spectrum of the phosphorylated protein with that of the non-phosphorylated protein. The appearance of additional resonances in the phosphorylated protein&#8217;s NMR spectrum confirms multiple phosphorylations.