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

Jana Klose; Norbert Wendt; Sybille Kubald; Eberhard Krause; Klaus Fechner; Michael Beyermann; Michael Bienert; Rainer Rudolph; Sven Rothemund

doi:10.1110/ps.04835904

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

Protein Sci. 2004 Sep;13(9):2470-5. doi: 10.1110/ps.04835904. Epub 2004 Aug 4.

Authors

Jana Klose¹, Norbert Wendt, Sybille Kubald, Eberhard Krause, Klaus Fechner, Michael Beyermann, Michael Bienert, Rainer Rudolph, Sven Rothemund

Affiliation

¹ Research Institute of Molecular Pharmacology, D-13125 Berlin, Germany.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Affinity Labels / chemistry
Affinity Labels / metabolism
Amino Acid Sequence
Animals
Corticotropin-Releasing Hormone / metabolism
Cysteine / chemistry
Disulfides / chemistry*
Histidine / chemistry*
Histidine / genetics
Histidine / metabolism
Molecular Sequence Data
Oligopeptides / chemistry*
Oligopeptides / genetics
Oligopeptides / metabolism
Protein Binding
Protein Folding*
Protein Structure, Tertiary
Rats
Receptors, Corticotropin-Releasing Hormone / chemistry*
Receptors, Corticotropin-Releasing Hormone / genetics
Receptors, Corticotropin-Releasing Hormone / metabolism*
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Sequence Alignment
Urocortins

Substances

Affinity Labels
CRF(2alpha) receptor
Disulfides
His-His-His-His-His-His
Oligopeptides
Receptors, Corticotropin-Releasing Hormone
Recombinant Proteins
Urocortins
corticotropin-releasing factor receptor type 2a, rat
Histidine
Corticotropin-Releasing Hormone
Cysteine