Spectroscopic studies on noncovalent binding of nicotinamide-modified BRCA1 (856-871) analogs to calf thymus DNA.

Various peptide drugs have entered the market with the development of molecular biology. Peptide drugs are used for treat diseases such as diabetes, breast cancer, and HIV infection. In this study, three nicotinamide-modified peptides were synthesized by modifying the N-terminus of BRCA1 (856-871, Y856R, K862Y, R866W) peptide with three nicotinic acid derivatives using solid-phase peptide synthesis. The results of calf thymus DNA (ctDNA) binding activity indicated that binding constants of BRCA1 (856-871, Y856R, K862Y, R866W) (P0) and three nicotinamide-modified peptides (P1, P2, and P3) to ctDNA were 1.89 × 103, 2.97 × 104, 7.61 × 104, and 8.09 × 104 L·mol-1, respectively. The binding affinity of the modified peptides was superior to that of BRCA1 (856-871, Y856R, K862Y, R866W). ΔHθ < 0 and ΔSθ < 0 indicated that van der Waals force and hydrogen bond contributed most to peptide-ctDNA binding. Results obtained by Circular dichroism (CD) indicated that peptide binding interaction led to conformational changes in ctDNA. Ultraviolet-visible (UV) spectroscopy, ethidium bromide (EB) competition experiments, DNA melting experiments, and viscosity measurements verified that peptides interacted with ctDNA via groove binding. Ionic strength experiments manifested that electrostatic binding was also involved in peptide-ctDNA binding.

Design, synthesis and interaction of BRC4 analogous peptides with RAD51(241-260).

Breast cancer susceptibility gene 2 (BRCA2) is an important tumor suppressor, which is participated in repair of damaged DNA by its highly conserved BRC repeat motifs regulating RAD51 protein homologous recombination and thereby preventing cell carcinogenesis. In this study, the BRCA2(1524-1548)-RAD51(241-260) complex structure was obtained based on PDB bank data 1N0W, which provided the basis for site-specific mutation of BRCA2(1524-1548). The BRC4 and BRC4 analogous peptides were synthesized, and the interaction between BRC peptide and RAD51(241-260) was studied by fluorescence spectroscopy, circular dichroism spectroscopy and microscale thermophoresis (MST). The results of circular dichroism showed that the changes in secondary structures of RAD51(241-260) occurred after adding BRC4 analogous peptides, and the α-helix content increased significantly. Fluorescence spectral data demonstrated that the model of BRC peptide binding to RAD51(241-260) was static quenching, and the binding constants of BRC4, P1, P2, P4 with RAD51(241-260) were 1.647 × 10-4 L mol-1, 2.532 × 10-4 L mol-1, 3.161 × 10-4 L mol-1, 1.705 × 10-4 L mol-1, respectively. The results of MST indicated that P2 and RAD51(241-260) have better affinity for dissociation constant 44.286 µM. The strongest affinity between P2 and RAD51(241-260) indicated that the mutation of amino acid residue constituting BRC α-helix affects the structure and interaction of BRC peptide and RAD51(241-260).

DNA/Lysozyme-binding affinity study of novel peptides from TAT (47-57) and BRCA1 (782-786) in vitro by spectroscopic analysis.

SISLL-TAT and TAT-SISLL were synthesized by modifying the N- or C-termini of cell-penetrating peptides as transacting activator of transcription TAT (47-57) by attaching BRCA1 (782-786) (SISLL). The novel peptides were synthesized through Fmoc solid-phase synthesis procedures and characterized by LCQ Fleet MS, 1H NMR and 13C NMR. SISLL-TAT and TAT-SISLL displayed forceful antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Salmonella typhimurium with low hemolysis. SISLL-TAT showed better antibacterial activity than TAT-SISLL, with the minimum inhibitory concentration (MIC) values of 10-33 µg·mL-1. The results of the DNA-binding activities showed that both SISLL-TAT and TAT-SISLL could interact with DNA via the minor groove mode, and the binding constants were 4.97 × 105 L·mol-1 and 4.42 × 105 L·mol-1 at 310 K, respectively. Circular dichroism analysis showed slight transformation of the lysozyme secondary structure caused by SISLL-TAT and TAT-SISLL. We also found that the novel peptides SISLL-TAT and TAT-SISLL targeted bacterial DNA resulting in cell death. This explains the antibacterial mechanism of SISLL-TAT and TAT-SISLL, and is a solid theoretical basis for further designing novel and highly effective antibiotics for clinical application.

Non-covalent interaction between CA-TAT and calf thymus DNA: Deciphering the binding mode by in vitro studies.

CA-TAT, a novel peptide analog, was modified at the N-terminus of TAT (47-57), the cell-penetrating peptide transacting activator of transcription, by attaching cecropin A (1-7). CA-TAT, TAT (47-57), and cecropin A (1-7) were synthesized using standard Fmoc solid-phase peptide synthesis procedures, purified using reversed-phase high performance liquid chromatography (RP-HPLC), and characterized using ESI-MS. CA-TAT demonstrated antibacterial activities against bacteria with low hemolysis (MHC > 128 µM). The minimum inhibitory concentration (MIC) values of CA-TAT were in the range of 1-16 µM, which completely inhibited both gram-positive and gram-negative bacteria. The interactions between CA-TAT or TAT (47-57) and calf thymus DNA (ct-DNA) were investigated using multi-spectroscopic techniques and viscometry. The results showed that both CA-TAT and TAT (47-57) can interact with DNA via the minor groove-binding mode, and binding constant was calculated to be 2.83 × 105 L mol-1 at 310 K, which is lower than that with the classical intercalation binder ethidium bromide (EB). Compared with TAT (47-57) or cecropin A (1-7), CA-TAT combined with DNA much closer. The study results suggest that CA-TAT can be used as a novel antibacterial peptide in the development of new antibiotics because of its antibacterial activity that targets intracellular DNA.