Jana Klose

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.

Influence of proline and β-turn mimetics on the cyclization of penta- and hexapeptides

Proline and Pro-derived peptidomimetics, such as meoxPro-Oic (4-methoxy-proline-octahydro indolic acid), and DBF (2-aminoethyl-6-dibenzofuran propionic acid) were introduced into thymopentin-derived penta- [SP5-] and hexa- [SP6-] peptides and penta-, hexa- and hepta-alanine. Surprisingly, we found that cyclomonomer formation in the investigated penta- and hexapeptides was drastically hindered by the presence of proline regardless of position.

2-Propanephosphonic acid anhydride (T3P)-mediated segment coupling and head-to-tail cyclization of sterically hindered peptides

In the course of comparing the effectiveness of an HOAt-derived coupling reagent (HAPyU) and a phosphonic acid-based condensation agent (T3P) by cyclization of model sequences, we found that T3P was a superior reagent with regard to conversion of the linear peptide into the stereochemically intact cyclic monomer when sterically hindered amino acids are found at the cyclization site.