Longer charged amino acids favor ß-strand formation in hairpin peptides.

Interactions between charged amino acids significantly influence the structure and function of proteins. The encoded charged amino acids Asp, Glu, Arg, and Lys have different number of hydrophobic methylenes linking the backbone to the charged functionality. It remains to be fully understood how does this difference in the number of methylenes affect protein structure stability. Protein secondary structures are the fundamental three-dimensional building blocks of protein structures. ß-Sheet structures are particularly interesting, because these structures have been associated with a number of protein misfolding diseases. Herein, we report the effect of charged amino acid side chain length at two ß-strand positions individually on the stability of a ß-hairpin. The charged amino acids include side chains with a carboxylate, an ammonium, or a guanidinium group. The experimental peptides, fully folded reference peptides, and fully unfolded reference peptides were synthesized by solid phase peptide synthesis and analyzed by 2D NMR methods including TOCSY, DQF-COSY, and ROESY. Sequence specific assignments were performed for all peptides. The chemical shift data were used to derive the fraction folded population and the folding free energy for the experimental peptides. Results showed that the fraction folded population increased with increasing charged amino acid side chain length. These results should be useful for developing functional peptides that adopt the ß-conformation.

Effect of lysine methylation and acetylation on the RNA recognition and cellular uptake of Tat-derived peptides.

The two lysine (Lys) residues in the human immunodeficiency virus trans-activator of transcription protein (HIV Tat protein) basic region (residues 47-57) are crucial for two bioactivities: RNA recognition and cellular uptake. Since the post-translational modifications of these two Lys residues affect the biological function of the Tat protein, we investigated the effect of methylation and acetylation of Lys50 and Lys51 in Tat-derived peptides on the two bioactivities. Tat-derived peptides, in which each lysine was replaced with a methylated- or acetylated-Lys, were synthesized by solid phase peptide synthesis. TAR RNA recognition of the peptides was studied by electrophoretic mobility shift assays. Cellular uptake of the peptides into Jurkat cells was determined by flow cytometry. Our results showed that acetylation of either Lys residue attenuated both bioactivities. In contrast, the effect of Lys methylation on the bioactivities depended on position and number of methyl groups. These findings should be useful for the development of functional molecules containing ammonium groups for RNA recognition to affect biological processes and for cellular uptake for drug delivery.