Scalable, Non-denaturing Purification of Phosphoproteins Using Ga3+-IMAC : N2A and M1M2 Titin Components as Study case
2019-04, Adams, Michael, Fleming, Jennifer R., Riehle, Eva, Zhou, Tiankun, Zacharchenko, Thomas, Markovic, Marija, Mayans, Olga
The purification of phosphorylated proteins in a folded state and in large enough quantity for biochemical or biophysical analysis remains a challenging task. Here, we develop a new implementation of the method of gallium immobilized metal chromatography (Ga3+-IMAC) as to permit the selective enrichment of phosphoproteins in the milligram scale and under native conditions using automated FPLC instrumentation. We apply this method to the purification of the UN2A and M1M2 components of the muscle protein titin upon being monophosphorylated in vitro by cAMP-dependent protein kinase (PKA). We found that UN2A is phosphorylated by PKA at its C-terminus in residue S9578 and M1M2 is phosphorylated in its interdomain linker sequence at position T32607. We demonstrate that the Ga3+-IMAC method is efficient, economical and suitable for implementation in automated purification pipelines for recombinant proteins. The procedure can be applied both to the selective enrichment and to the removal of phosphoproteins from biochemical samples.
CARP interacts with titin at a unique helical N2A sequence and at the domain Ig81 to form a structured complex
2016-09, Zhou, Tiankun, Fleming, Jennifer R., Franke, Barbara, Bogomolovas, Julius, Barsukov, Igor, Rigden, Daniel J., Labeit, Siegfried, Mayans, Olga
The cardiac ankyrin repeat protein (CARP) is up-regulated in the myocardium during cardiovascular disease and in response to mechanical or toxic stress. Stress-induced CARP interacts with the N2A spring region of the titin filament to modulate muscle compliance. We characterize the interaction between CARP and titin-N2A and show that the binding site in titin spans the dual domain UN2A-Ig81. We find that the unique sequence UN2A is not structurally disordered, but that it has a stable, elongated α-helical fold that possibly acts as a constant force spring. Our findings portray CARP/titin-N2A as a structured node and help to rationalize the molecular basis of CARP mechanosensing in the sarcomeric I-band.