title: NMR characterization of foldedness for the production of E3 RING domains.
abstract: We summarize the use of NMR spectroscopy in the production and the screening of stability and foldedness of protein domains, and apply it to the RING domains of E3 ubiquitin-ligases. RING domains are involved in specific interactions with E2 ubiquitin-conjugating enzymes and thus play an essential role in the ubiquitination pathway. Protein production of the Zn(2+) containing and cysteine rich RING domains for molecular studies frequently turns out to be problematic. We compared the expression and solubility of 14 E3 RING/U-box domains fused to the N-terminal tags of His(6), His(6)-GB1, His(6)-Trx and His(6)-GST at small scale and analyzed, by NMR spectroscopy, their correct folding after purification. The addition of GST, Trx or GB1 to the N-terminal His(6) tag significantly improved both the expression and solubility of target proteins as compared to His(6) tag alone. More importantly most of the immobilized metal affinity chromatography (IMAC) purified proteins were largely unfolded as judged by analysis of the (1)H-(15)N HSQC spectra. We demonstrate that imidazole causes a concentration dependent decrease in stability of RING proteins ascribed to metal depletion and resulting in unfolding or precipitation. In contrast, using glutathione affinity chromatography, the His(6)-GST fused RING and U-box domains were purified as correctly folded proteins with high yields. Our data clearly demonstrate that IMAC should be avoided and that GST-fusion affinity chromatography is generally applicable for expression and purification of Zn(2+) containing proteins.
authors.affiliation: Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, Utrecht, The Netherlands.
authors.forename: Anding
authors.initials: A
authors.lastname: Huang
authors.forename: Rob N
authors.initials: RN
authors.lastname: de Jong
authors.forename: Gert E
authors.initials: GE
authors.lastname: Folkers
authors.forename: Rolf
authors.initials: R
authors.lastname: Boelens
mesh_terms: D002846:Chromatography, Affinity; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D017510:Protein Folding; D055550:Protein Stability / Q000187:drug effects; D054829:RING Finger Domains; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D007093:Imidazoles; D011993:Recombinant Fusion Proteins; D006639:Histidine; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D015032:Zinc; D003545:Cysteine