Ubiquitin binds to a short peptide segment of hydrolase UCH-L3: a study by FCS, RIfS, ITC and NMR.
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 muM 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 beta 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.
Roth
Günter
G
Institut für Organische Chemie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany.
Freund
Stefan
S
Möhrle
Bernd
B
Wöllner
Karin
K
Brünjes
Jente
J
Gauglitz
Günter
G
Wiesmüller
Karl-Heinz
KH
Jung
Günther
G
D000595:Amino Acid Sequence; D002151:Calorimetry; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D007368:Interferometry; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D043222:Ubiquitin Thiolesterase
D008024:Ligands; D010455:Peptides; D025801:Ubiquitin; C509806:UCHL3 protein, human; D043222:Ubiquitin Thiolesterase; D003546:Cysteine Endopeptidases