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

Abstract

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.

The “Recognition Helix” of the Type II Acyl Carrier Protein (ACP) Utilizes a “Ubiquitin Interacting Motif (UIM)”-like Surface To Bind Its Partners

“…In the yeast transcription factor Met4, the first conserved hydrophobic residue is replaced with a “Glu”, and in AtDAR1 UIM1, the same residue is replaced with a “Cys”. In ubiquitin carboxyl-terminal hydrolase isozyme L3, the UIM motif lacks any carboxylates at the C-terminus . The Gα s UIM motif is shorter than usual, lacking the carboxylates at the N-terminus .…”

“…In light of the noncanonical UIMs, and our studies, the hydrophobic element -ϕ-x-x-Ala-ϕ - appears to be the most crucial component of UIM, flanked by negatively charged residues on either side. The hydrophobic element can vary from -ϕ-x-Ala-ϕ - as in HsGα s UIM to -ϕ-x-x-x-Ala-ϕ - as in HsUCHL-3 . In the acidic transactivation domain of erythroid-specific transcription factor EKLF IUIM, this hydrophobic sequence is replaced with WWKLF, and the peptide binds ubiquitin .…”

Mode of Ezrin-Membrane Interaction as a Function of PIP 2 Binding and Pseudophosphorylation

“…RIfS is an optical technique that allows label-free investigations of protein binding to a specific receptor lipid embedded in SLBs (27,(31)(32)(33)(34)(35). The adsorption process is detected as an increase in optical thickness, OT ¼ n d, i.e., the product of the refractive index n and the physical layer thickness d. RIfS experiments were employed to analyze whether ezrin wt or ezrin T567D adsorbed to either the receptor lipid DOGS-Ni-NTA or PIP 2 results in a difference in OT.…”

Structural Oscillations of Non–muscle Myosin II–C2: Time Resolved Confocal Microscopy

“…Confocal imaging and FCS have recently been applied to unravel many biochemical phenomena, such as, gene expression at the single molecule level, single molecule enzymatic reaction, aggregation of the polyQ proteins with other cellular proteins, and intermittent structural oscillation of different organelles in a live cell . He et al., have demonstrated the open‐close dynamics of the enzyme HPPK which gives rise to intermittent coherence.…”