Details

Autor(en) / Beteiligte

• Roth, Günter
• Freund, Stefan
• Möhrle, Bernd
• Wöllner, Karin
• Brünjes, Jente
• Gauglitz, Günter
• Wiesmüller, Karl-Heinz
• Jung, Günther

Titel

Ubiquitin Binds to a Short Peptide Segment of Hydrolase UCH-L3: A Study by FCS, RIfS, ITC and NMR

Ist Teil von

• Chembiochem : a European journal of chemical biology, 2007-02, Vol.8 (3), p.323-331

Ort / Verlag

Weinheim: Wiley-VCH Verlag

Erscheinungsjahr

2007

Link zum Volltext

Quelle

Wiley Blackwell Single Titles

Beschreibungen/Notizen

• Screening for small peptidic affinity tags for the detection of ubiquitin and ubiquitinated proteins yielded the dodecapeptide amide DPDELRFNAIAL-NH₂ as a specific ubiquitin-interacting ligand. A peptide collection--based on crystal structures with ubiquitin-interacting proteins--was designed and confirmed by sequence comparison of ubiquitin-interacting motifs. Four independent physical detection methods demonstrated that the peptide binds to monomeric ubiquitin with an affinity of about 10 μM and with fast on and off rates. Fluorescence correlation spectroscopy with fluorescent peptides showed specific interaction with ubiquitin. Reflectometric interference spectroscopy with surface-immobilized peptides and isothermal calorimetry measurements confirmed the specific binding of ubiquitin and fast rate constants. ¹H,¹⁵N heteronuclear NMR localised the interaction site across the β sheet of ubiquitin. The peptide aligns well with the ubiquitin-interacting motif and represents a lead structure for the rational design of high-affinity tags for targeting ubiquitinated protein in vitro and in vivo.