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The nuclear localization signal sequence (NLS) of the transcription factor NF- κB is a cationic peptide with the ability to cross the cytoplasmic membrane and facilitate the delivery of attached cargo, such as DNA and proteins, to cells. Previous research had pointed to the essential role of cationic residues, therefore, the importance of residues within the NLS of NF- κB was evaluated for cellular uptake using an alanine replacement strategy. Although it was expected that removal of the cationic groups would have the greatest effect on membrane translocation, the most significant decreases in cellular uptake occurred with the replacement of the hydrophilic Q6 (80%) and the hydrophobic L8 (70%) residues. Replacement of the positively charged residues resulted in 30–40% decrease in cellular uptake, indicating that electrostatic interactions are not the primary driving force for membrane translocation. [ABSTRACT FROM AUTHOR]

Abstract: A nonphosphorylated disulfide-bridged peptide, cyclo(Cys-Glu[sup 1]-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr[sup 9]-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC[sub 50] of 15.5 µm) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side-chains of amino acids Glu[sup 1], Tyr[sup 3], Glu[sup 4], Asn[sup 5], and Met[sup 8]. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a β-turn-like conformation. Replacement of Tyr[sup 3] or Asn[sup 5] with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu[sup 1], i.e. the amino acid N-terminal to Y[sup 3], remarkably reduces the binding affinity. The position of the Glu[sup 1] side-chain is confirmed to provide a complementary role for pTyr[sup 3], as demonstrated by the low micromolar inhibitory activity (IC[sub 50] = 1.02 µm) of the nonphosphorylated peptide 11, G1(Gla[sup 1]), in which Glu[sup 1] was replaced by γ-carboxy-glutamic acid (Gla). [ABSTRACT FROM AUTHOR]