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.
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.
Klose
Jana
J
Research Institute of Molecular Pharmacology, D-13125 Berlin, Germany.
Wendt
Norbert
N
Kubald
Sybille
S
Krause
Eberhard
E
Fechner
Klaus
K
Beyermann
Michael
M
Bienert
Michael
M
Rudolph
Rainer
R
Rothemund
Sven
S
D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D054832:Urocortins
D000345:Affinity Labels; C101518:CRF(2alpha) receptor; D004220:Disulfides; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; D054832:Urocortins; C499434:corticotropin-releasing factor receptor type 2a, rat; D006639:Histidine; D003346:Corticotropin-Releasing Hormone; D003545:Cysteine