Structural characterization of a mutant peptide derived from ubiquitin: Implications for protein folding

The formation of the N-terminal beta -hairpin of ubiquitin is thought to be an early event in the folding of this small protein. Previously, we have s hown that a peptide corresponding to residues 1-17 of ubiquitin folds auton omously and is likely to have a native-like hairpin register. To investigat e the causes of the stability of this fold, we have made mutations in the a mino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U(1-17)T9D, the native conformation is stabilized with re spect to the wild-type sequence, so much so that we are able to characteriz e the structure of the mutant peptide fully by NMR spectroscopy. The data i ndicate that U(1-17)T9D peptide does indeed form a hairpin with a native-li ke register and a type I turn with a G1 beta -bulge, as in the full-length protein. The reason for the greater stability of the U(1-17)T9D mutant rema ins uncertain, but there are nuclear Overhauser effects between the side ch ains of Asp9 and Lys11, which may indicate that a charge-charge interaction between these residues is responsible.