Purification of a single cysteine Arrestin3 (Arr3) variant for 19F NMR study

Abstract

Non-visual arrestins (arrestin 2 & 3) scaffold mitogen-activated protein kinase (MAPK) cascades. Although all three canonical MAPK families are mediated by arrestin3, the structural basis of these interactions remain largely unknown. In order to study the potential binding-induced conformational changes of arrestin3 by MAP kinases, we developed a thio-based 19F-NMR by incorporating 19F into cysteine residue on arrestin3 by covalent-modification. We first have to construct and isolate the arrestin3 variant with single cysteine residue. Here we reported our initial effort to purify a single cysteine arrestin3 variant (Arr3-V8C) by using Heparin-sepharose, Q-sepharose and SP-sepharose.