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Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a

¹ Research Institute of Molecular Pharmacology, D-13125 Berlin, Germany
² Martin-Luther University Halle-Wittenberg, D-01620 Halle, Germany
³ Interdisciplinary Center for Clinical Research, D-04103 Leipzig, Germany

(RECEIVED April 23, 2004; FINAL REVISION May 17, 2004; ACCEPTED May 17, 2004)

The oxidative folding, particularly the arrangement of disulfide bonds of recombinant extracellular N-terminal domains of the corticotropin-releasing factor receptor type 2a bearing five cysteines (C2 to C6), was investigated. Depending on the position of a His-tag, two types of disulfide patterns were found. In the case of an N-terminal His-tag, the disulfide bonds C2–C3 and C4–C6 were found, leaving C5 free, whereas the C-terminal position of the His-tag led to the disulfide pattern C2–C5 and C4–C6, and leaving C3 free. The latter pattern is consistent with the disulfide arrangement of the extracellular N-terminal domain of the corticotropin-releasing factor (CRF) receptor type 1, which has six cysteines (C1 to C6) and in which C1 is paired with C3. However, binding data of the two differently disulfide-bridged domains show no significant differences in binding affinities to selected ligands, indicating the importance of the C-terminal portion of the N-terminal receptor domains, particularly the disulfide bond C4–C6 for ligand binding.

Keywords: protein folding; affinity tag; corticotropin-releasing factor receptor

Abbreviations: ACN, acetonitrile • BSA, bovine serum albumin • CDAP, cyanodimethylaminopyridinium tetrafluoroborate • CRF, corticotropin-releasing factor • DTT, dithiothreitol • GPCR, G protein–coupled receptor • GST, glutathione S-transferase • GuHCl, guanidinium hydrochloride • HPLC, high-performance liquid chromatography • IAA, iodoacetamide • MALDI, matrix-assisted laser desorption/ionization • MBP, maltose binding protein • MS, mass spectrometry • NT, N terminus • RP, reversed-phase • SPA, scintillation proximity assay • TOF, time of flight

Reprint requests to: Sven Rothemund, Interdisciplinary Center for Clinical Research, Inselstrasse 22, D-04103 Leipzig, Germany, e-mail: sven_r{at}yahoo.com; fax: +49-341-97-+5979; or Jana Klose, Research Institute of Molecular Pharmacology, Robert-Roessle-Str. 10, D-13125 Berlin, Germany, e-mail: jana_klose{at}yahoo.com; fax: +49-30-94793-159.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04835904.

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