@article{THESIS,
      recid = {718},
      author = {Henderson, James Michael},
      title = {Antimicrobial Peptides' Selectivity and Line Activity  Governed by Membrane Properties},
      publisher = {University of Chicago},
      school = {Ph.D.},
      address = {2016-06},
      number = {THESIS},
      pages = {200},
      abstract = {As the number of multidrug resistant pathogens rise, the  search for new antibiotics is crucial. Antimicrobial  peptides (AMPs) have received attention to fill this  pharmacological void as these small host defense molecules  induce selective membrane lytic activity against microbial  pathogens. The ability of AMPs to target the microbial  membrane over that of a host’s has naively been based on  the electrostatic attraction of these predominately  cationic peptides for the negatively charged microbial  membrane. Despite several decades of intense research their  application as therapeutic treatments have been hampered  from a clear understanding of their universal mechanism of  interaction, given these molecular species vary widely in  terms of both their primary sequence and secondary  structures making structure-function relationships  difficult.
We have previously shown with atomic force  microscopy (AFM) that zwitterionic membranes display  concentration-dependent structural transformations induced  by Protegrin-1 (PG-1)—an 18-residue, cationic, β-sheet AMP  isolated from pig leukocytes—that display finger-like  instabilities at bilayer edges, to the formation of  transmembrane pores, and finally to a network of worm-like  micelles. The peptide-induced disruption beyond pores of a  static size has suggested that AMPs act to lower the  interfacial energy of the bilayers in a way similar to  detergents. The increasing degree of membrane disruption in  charge-neutral membranes demonstrates that a more complex  interaction than that suggested by a simple electrostatic  argument is needed to explain AMP selectivity in general. I  have proposed that in addition to an electrostatic element,  specific membrane compositional differences between host  and pathogen tunes AMP activity to selectively disrupt  microbial membranes. The presence of cholesterol in  eukaryotic cell membranes is one of the crucial differences  between host and bacterial cell membranes, which evince  none. Given that cholesterol stiffens fluid membranes, AMP  selectivity may in part be the result of differing membrane  fluidities. Isothermal titration calorimetry (ITC)  measurements were conducted to characterize PG-1’s affinity  to vesicles with increasing cholesterol percentages and has  shown decreasing insertion which is ultimately negated by  30 mole% cholesterol. Corollary AFM imaging has supported  that PG-1 is less able to insert into  cholesterol-containing membranes, as a reduction in pore  density and overall disruption was observed. Contrastingly,  X-ray grazing-incidence diffraction measurements show that  increasing monolayer fluidity, through disruption of the  packing of rigid lipid monolayers, can instead enhance  PG-1’s insertion, confirming PG-1’s behavior is driven by  the mechanical response of the membrane.
Cholesterol’s  unique ability to impart greater membrane cohesion results  in thicker membranes that potentially affects the  hydrophobic matching between membrane and peptide, reducing  favorable peptide insertion that makes the membrane more  resistive. Through oriented circular dichroism studies we  effectively showed that when PG-1 was pre-incorporated into  mixed membranes containing cholesterol, PG-1’s membrane  orientation was quite plastic and indeed confirmed  hydrophobic matching can be a regulating factor in PG-1’s  activity. Combined AFM and ITC measurements have shown that  given membranes of the same relative fluidity, membranes  composed of lipids with longer acyl chain lengths are more  resistant to the action of PG-1.
Through the study of  thirteen AMPs that differ in charge, sequence, and  secondary structure from across a variety of living  species, we show that a common physical ability for the  peptides to lower line tension unites their interaction  with membranes. While the line-active behavior was not  driven by the overall charge of the peptide, the observed  line activity was largely correlated with the adoption of  imperfect secondary structures, generated by poor  amphiphilic residue segregation or breaks in ideal  secondary motifs, that commonly positioned charged residues  near the membrane interface to favorably induce membrane  deformation. The observed universal detergent-like behavior  for AMPs makes a fluidity argument for selectivity much  more general for AMPs of all charges and potentially  implicates other physical attributes of membrane, e.g.,  lipid spontaneous curvature, in regulating AMP activity.},
      url = {http://knowledge.uchicago.edu/record/718},
      doi = {https://doi.org/10.6082/M1FT8J34},
}