Structural Regulation of a Neurofilament-Inspired Intrinsically Disordered Protein Brush by Multisite Phosphorylation

• DOI: 10.1021/acs.biochem.8b00007
• Publication Date: 2018-03-20T14:09:20Z
• Authors: Ruoxing Lei; Jessica P. Lee; Matthew B. Francis; Sanjay Kumar
• Keywords: Intrinsically Disordered Proteins; Dephosphorylation; Hyperphosphorylation; Phosphorylation cascade

Intrinsically disordered proteins (IDPs) play central roles in numerous cellular processes. While IDP structure and function are often regulated by multisite phosphorylation, the biophysical mechanisms linking these post-translational modifications to remain elusive. For example, intrinsically C-terminal sidearm domain of neurofilament heavy subunit (NFH-SA) forms a dense brush along axonal NF backbones is subject extensive serine phosphorylation. Yet, insight into relationship between phosphorylation has been limited lack paradigms which conformational responses can be measured setting controlled Here, we approach this question immobilizing recombinant NFH-SA (rNFH-SA) as brushes onto glass, controllably phosphorylating sequence situ with mitogen-activated protein kinase 1 (ERK2) preactivated (MKK). We then monitor height changes using atomic force microscopy, shows that induces significant swelling an extent strongly depends upon pH ionic strength, consistent mechanism regulates through local electrostatic interactions. Further mechanism, phosphorylated rNFH-SA may dramatically condensed micromolar concentrations divalent cations. Phosphorylation-induced qualitatively reversible via alkaline phosphatase-mediated dephosphorylation. Our study demonstrates controls modulation chain electrostatics points general strategy for engineering IDP-based interfaces reversibly dynamically modulated enzymes.