Design and Optimization of a Cholesterol-Binding Peptide Based on the Cholesterol Recognition Amino acid Consensus Motif
Advisor: Angela Brown
Numerous bacterial toxins bind cholesterol on the host cell membrane as a first step in recognition of the cell.This research focuses on the repeats-in-toxin (RTX) protein, leukotoxin A (LtxA), which acts by disrupting the host’s immune response by eliminating nearby leukocytes in a cholesterol-dependent mechanism. LtxA uses a cholesterol recognition amino acid consensus (CRAC) motif to bind cholesterol with a strong affinity. Our lab has used the CRAC sequence of LtxA to design a cholesterol-binding peptide to inhibit bacterial toxin binding to host cells. We are currently working to optimize the affinity of the peptide for cholesterol by varying the charge and hydrophobicity of the peptide sequence.
We varied the CRAC peptide sequence by presenting conservative mutations of charged residues in the flanking domain of CRACWT to synthesize CRAC-1F. We hypothesized that reducing the charge of the peptide would enhance its partitioning into the membrane and therefore improve its affinity for cholesterol. Using surface plasmon resonance (SPR), we demonstrated that the peptide has a strong, but reduced affinity, for cholesterol relative to CRACWT. However, we demonstrated that the CRAC-1F peptide effectively inhibited binding of LtxA to cholesterol in giant unilamellar vesicle (GUV) membranes. As a result of this inhibition of cholesterol binding, the peptide was able to inhibit LtxA-mediated cytotoxicity. These findings indicate that increasing membrane partitioning alone will not enhance cholesterol-binding by these peptides. This work represents an important step in understanding the structural context of improving the efficiency of peptide-mediated inhibition of LtxA binding to cholesterol by changing the peptide’s primary structure. Our goal was to discover the impact that these conservative mutations will have on the peptide’s affinity for cholesterol. We are currently building on these results by designing and testing additional CRAC peptides with conservative mutations to observe the impact on cholesterol affinity and inhibition of toxin activity.
About Angela Sinani:
Angela Sinani is currently a junior at Lehigh University pursuing a Bioengineering Bachelor degree with a focus in biomechanics and biomaterials. Angela is a member of a number of professional and student organizations including the Lehigh Chapter of the Biomedical Engineering Institute (Student Society of Biomedical Engineers), Biomedical Engineering Society (BMES) and Women in Engineering.
At Lehigh, Angela has been involved in academic research for the past two years by focusing on antibiotic resistance. Angela’s undergraduate research, under Professor Brown, consists of identifying therapeutic drug targets to prevent disease pathogenesis and reciprocally, use this approach in order to develop therapeutics agents that block the function of the cell penetration mechanisms. Angela proudly presented her research findings under Professor Brown’s guidance in the annual Biomedical Engineering Society Conference 2017 in Phoenix Arizona.
During the past summer, Angela participated in bioengineering research internship with the Howard Hughes Medical Institute, a nationally renowned non-profit medical research organization. This experience provided her with valuable experience and insight into hypothesis development, experimental design, and result presentation.