Transmembrane pore formation by the carboxyl terminus of Bax protein.
Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal "helix 9" of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide-peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents.
Department of Physics, College of Sciences, University of Central Florida, Orlando, FL, USA; Biotechnology Graduate Program, College of Medicine, University of Central Florida, Orlando, FL, USA.
Pranav
P
Garg
Kathleen N
KN
Nemec
Annette R
AR
Khaled
Suren A
SA
Tatulian
D000595:Amino Acid Sequence; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D051336:Mitochondrial Membranes / Q000378:metabolism; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013997:Time Factors; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000737:chemistry; D051028:bcl-2-Associated X Protein / Q000737:chemistry*
C494772:BAK1 protein, human; C494826:BAX protein, human; D005452:Fluoresceins; D010455:Peptides; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D045304:Cytochromes c; D020169:Caspases; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; C007740:fluorexon