title: Insoluble protein characterization by circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR).
abstract: Besides misfolded proteins, which still retain the capacity to fold into uniquely defined structures but are misled to "off-pathway" aggregation, there exists a group of proteins which are unrefoldable and insoluble in buffers. Previously no general method was available to solubilize them and consequently their solution conformations could not be characterized. Recently, we discovered that these insoluble proteins could in fact be solubilized in pure water. Circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) characterization led to their classification into three groups, all of which lack the tight tertiary packing and consequently anticipated to unavoidably aggregate in vivo with ~150 mM ions, thus designated as "intrinsically insoluble proteins (IIPs)." It appears that eukaryotic genomes contain many "IIP," which also have a potential to interact with membranes to trigger neurodegenerative diseases. In this chapter, we provide a detailed procedure to express and purify these proteins, followed by CD and NMR spectroscopy characterization of their conformation and interaction with dodecylphosphocholine (DPC).
authors.affiliation: Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119260, Singapore.
authors.forename: Shaveta
authors.initials: S
authors.lastname: Goyal
authors.forename: Haina
authors.initials: H
authors.lastname: Qin
authors.forename: Liangzhong
authors.initials: L
authors.lastname: Lim
authors.forename: Jianxing
authors.initials: J
authors.lastname: Song
mesh_terms: D000818:Animals; D002942:Circular Dichroism / Q000379:methods; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins; D010767:Phosphorylcholine; C028810:dodecylphosphocholine