Concerns Regarding the Practice of Multiple Detector Size-Exclusion Chromatography

1995 ◽  
pp. 59-68 ◽  
Author(s):  
Christian Jackson ◽  
Howard G. Barth
Keyword(s):  
Size Exclusion Chromatography ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

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