Characterization of large peptide fragments derived from the N-terminal domain of the ribosomal protein L9: Definition of the minimum folding motif and characterization of local electrostatic interactions

A set of peptides derived from the N-terminal domain of the ribosomal protein L9 (NTL9) have been characterized in an effort to define the minimum unit of this domain required to fold and to provide model peptides for the analysis of electrostatic interactions in the unfolded state. NTL9 is a 56-residue α-β protein with a β1-loop-β2-α1-β3-α2 topology. The β-sheet together with the first helix comprise a simple example of a common supersecondary motif called the split β-α-β fold. Peptides corresponding to the β1-loop-β2 unit are unstructured even when constrained by an introduced disulfide. The pK_as of Asp-8 and Glu-17 in these peptides are slightly lower than the values found for shorter peptides but are considerably higher than the values in NTL9. A 34-residue peptide, which represents the β1-loop-β2-α1 portion of NTL9, is also unstructured. In contrast, a 39-residue peptide corresponding to the entire split β-α-β motif is folded and monomeric as judged by near- and far-UV CD, two-dimensional NMR, ANS binding experiments, pK_a measurements, and analytical ultracentrifugation. The fold is very similar to the structure of this region in the intact protein. Thermal and urea unfolding experiments show that it is cooperatively folded with a δG° of unfolding of 1.8-2.0 kcal/mol and a T_m of 58 °C. This peptide represents the first demonstration of the independent folding of an isolated split β-α-β motif, and is one of only four naturally occurring sequences of fewer than 40 residues that has been shown to fold cooperatively in the absence of disulfides or ligand binding.