Biochemical and Biophysical Research Communications
A ubiquitin-interacting motif from Hrs binds to and occludes the ubiquitin surface necessary for polyubiquitination in monoubiquitinated proteins
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Materials and methods
Sample preparation. [U-13C, 15N]ubiquitin was produced using a pET3a (Novagen) plasmid, which was a gift of Dr. C.M. Pickart. The plasmid containing a gene for human ubiquitin was transformed into BL21(DE3) cell line and grown at 37 °C on M9 media supplemented with [13C]glucose and 15NH4Cl as sole sources of carbon and nitrogen. Protein expression was induced by adding 1 mM IPTG once A600 reached 0.6. The cells were then grown at 30 °C overnight, harvested by centrifugation (5000 g for 20 min), and
A well-defined helical structure encompasses the hydrophobic central part of the UIM_Hrs peptide
Secondary structure prediction programs from the PredictProtein server [27] indicate that amino acids from the hydrophobic part of the peptide have a predisposition to form α-helix. A CD spectrum of the UIM_Hrs dissolved in water indicated that UIM_Hrs has 45% α-helix (Fig. 1A). To characterize the secondary structure of the UIM_Hrs at the residue specific level, we performed two-dimensional homonuclear experiments, [1H–1H] TOCSY and [1H–1H] NOESY (data not shown). Sequential assignments of
Acknowledgements
This work was supported by NIH Grant GM 47021 and NCI Fellowship F037244.
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