Conformational Fine- Tuning of Pore- Forming Peptide Potency and Selectivity

Transcription

1 Conformational Fine- Tuning of Pore- Forming Peptide Potency Aram J. Krauson, O. Morgan Hall, Taylor Fuselier, Charles G. Starr, W. Berkeley Kauffman, and William C. Wimley, J. Am. Chem. Soc. 2015, 137, Ian Crouch Burke Lab March 5 th, 2016

2 Membrane- Permeablizing Peptides one β- barrel subunit of the trimeric OmpF potassium channel α- helix tetramer KcsA Melittin 26- aa peptide component of bee venom cycloviolacin O 2 cyclic peptide

3 Membrane Pore- Forming Peptides Chemosensitizing cyclic peptides: Antimicrobial short lipopeptides: drug- resistant breast cancer cells cycloviolacin O 2 + doxorubicin + STYOX stain (green) 0 min. 15 min. 30 min. Gerlach, S. L.; Rathinakumar, R.; Chakravarty, G.; Goransson, U.; Wimley, W. C.; Darwin, S. P.; Mondal, D. Biopolymers 2010, 94, Makovitzki, A.; Avrahami, D.; Shai, Y. Proc. Natl. Acad. Sci. U. S. A. 2006, 103,

4 Melittin 26 residue α- helix separated into 2 segments by proline, giving rise to a dynamic, disordered pore state. cationic (+6) C- terminal segment promotes binding to negatively charged phospholipids amphipathic α- helix with a nonpolar surface that drives partitioning into lipid bilayer membranes pore properties vary greatly depending on ph, temperature, ionic strength, concentration, and lipid identity A diastereomeric melittin containing D- amino acids has a lower tendency to form α- helices and so does not lyse mammalian cells but retains good antibacterial activity. Shai, Y.; Oren, Z. Peptides 2001, 22, Increased antibacterial activitywas obtianed from a fusion peptide combining melittinwith cecropin A, an antibacterial peptide from insects. Oh, D.; Shin, S. Y.; Kang, J. H.; Hahm, K. S.; Kim, K. L.; Kim, Y. J. Pept. Res. 1999, 53, Template- assembledtetramers of melittinhave potent pore- forming activity. Pawlak, M.; Meseth, U.; Dhanapal, B.; Mutter, M.; Vogel, H. Protein Sci. 1994, 3, Melittin molecules incorporated into targeted nanoparticles ( nanobees ) have anticancer activity. Pan, H.; Soman, N. R.; Schlesinger, P. H.; Lanza, G. M.; Wickline, S. A. Wiley. Interdiscip. Rev. Nanomed. Nanobiotechnol. 2011, 3,

5 Melittin 26 residue α- helix separated into 2 segments by proline, giving rise to a dynamic, disordered pore state. cationic (+6) C- terminal segment promotes binding to negatively charged phospholipids amphipathic α- helix with a nonpolar surface that drives partitioning into lipid bilayer membranes pore properties vary greatly depending on ph, temperature, ionic strength, concentration, and lipid identity A diastereomeric melittin containing D- amino acids has a lower tendency to form α- helices and so does not lyse mammalian cells but retains good antibacterial activity. Shai, Y.; Oren, Z. Peptides 2001, 22, Increased antibacterial activitywas obtianed from a fusion peptide combining melittinwith cecropin A, an antibacterial peptide from insects. Oh, D.; Shin, S. Y.; Kang, J. H.; Hahm, K. S.; Kim, K. L.; Kim, Y. J. Pept. Res. 1999, 53, Template- assembledtetramers of melittinhave potent pore- forming activity. Pawlak, M.; Meseth, U.; Dhanapal, B.; Mutter, M.; Vogel, H. Protein Sci. 1994, 3, Melittin molecules incorporated into targeted nanoparticles ( nanobees ) have anticancer activity. Pan, H.; Soman, N. R.; Schlesinger, P. H.; Lanza, G. M.; Wickline, S. A. Wiley. Interdiscip. Rev. Nanomed. Nanobiotechnol. 2011, 3,

6 Pore formation by anti- microbial peptides (AMPs) A. Barrel- stave model B. Carpet (detergent- like) model C. Toroidal- pore model

7 Pore formation by melittin A. Barrel- stave model B. Carpet (detergent- like) model C. Toroidal- pore model electron density melittin di18:0 (9,10Br)PC mixture (1:40 P/L) Lee, M. T., Sun, T. L., Hung, W. C., & Huang, H. W. (2013) Proceedings of the National Academy of Sciences, 110(35),

8 combinatorial library of 7776 (2 5 x 3 5 ) melittin analogs varied residues were chosen to test specific hypotheses Peptides were screened for loss- of- function using POPC LUVs at a higher peptide to lipid ratio than the ratio at which melittin permeablizes the same LUVs.

9 Two- step screen for permeablization by melittinanalogs: Step 1: Step 2: (lipid) O N + O - NH NBD dye N O N dithionite (K 2 S 2 O 4 ) added at equilibrium NBD dye reduction

10

11 function- retaining peptides loss- of- function peptides

12

13 MelP5 = gain of function peptide from earlier work MelN2 = loss- of- function from library Mel L16G = loss- of- function single AA change

14 MelP5 = gain of function peptide from earlier work MelN2 = loss- of- function from library Mel L16G = loss- of- function single AA change

15 MelP5 = gain of function peptide from earlier work MelN2 = loss- of- function from library Mel L16G = loss- of- function single AA change

16 random coils or helices in solution POPC bilayer K 1 K 2 surface-bound helices toroidal pore formation

17 random coils or helices in solution POPC bilayer K 1 K 2 surface-bound helices toroidal pore formation K 1(Mel L16G), K 1(Mel N2) < K 1(melittin) K 2(Mel L16G), K 2(Mel N2), K 2(melittin) < K 2(Mel P5)