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Ubiquitin Binds to a Short Peptide Segment of Hydrolase UCH‐L3: A Study by FCS, RIfS, ITC and NMR
Author(s) -
Roth Günter,
Freund Stefan,
Möhrle Bernd,
Wöllner Karin,
Brünjes Jente,
Gauglitz Günter,
Wiesmüller KarlHeinz,
Jung Günther
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600254
Subject(s) - isothermal titration calorimetry , ubiquitin , chemistry , peptide , target peptide , biochemistry , biophysics , nuclear magnetic resonance spectroscopy , stereochemistry , biology , gene
Screening for small peptidic affinity tags for the detection of ubiquitin and ubiquitinated proteins yielded the dodecapeptide amide DPDELRFNAIAL‐NH 2 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 μ M 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. 1 H, 15 N 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.