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Investigation of the thermodynamics of DNA interactions with the LL‐37 peptide
Author(s) -
Dodson CarolAnn Elizabeth,
Zachary Ralph Karl,
Craig Maria Lee
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.740.2
Subject(s) - isothermal titration calorimetry , peptide , dna , cpg site , chemistry , ethidium bromide , microbiology and biotechnology , electrophoretic mobility shift assay , innate immune system , tlr9 , biophysics , biochemistry , receptor , biology , dna methylation , gene , gene expression
Previous studies of LL‐37, an antimicrobial peptide of innate immunity, show that LL‐37 has effects in lupus models and in breast and prostate cancer. This peptide is able to bind to DNA and diffuse through the lipid bi‐layer of cells, allowing entry of self‐DNA that can upregulate the immune system through Toll‐like receptor 9 (TLR9). In prostate and breast cancer, this small peptide becomes increasingly over‐expressed in tumor tissue as the cancer progresses. To further understand the biochemical nature of the DNA/LL‐37 complex, we have used electrophoretic mobility shift assays (EMSA) and isothermal titration calorimetry (ITC) to determine thermodynamic parameters, including ΔH, K d , and peptide:DNA ratio, of complex formation between LL‐37 and CpG DNA sequences known to activate TLR9. By quantitative analysis of EMSA results, we observe a decrease in ethidium bromide staining when the concentration of LL‐37 is increased beyond that needed to shift all DNA and conclude that increasing LL‐37 concentrations leads to additional deposition of LL‐37 on the DNA. ITC results provide evidence of a stable complex between LL‐37 and CpG DNA. Future directions include studies of small molecules capable of blocking the binding of the peptide to the DNA. This work was supported by a Virginia Foundation of Independent Colleges Summer Undergraduate Research Fellowship.