title: Photocontrollable peptide-based switches target the anti-apoptotic protein Bcl-xL.
abstract: Photocontrol of Bcl-x(L) binding affinity has been achieved by using short BH3 domain peptides for Bak(72-87) and Bid(91-111) alkylated with an azobenzene crosslinker through two cysteine residues with different sequence spacings. The power to control the conformation of the crosslinker and hence peptide structure was demonstrated by CD and UV/Vis spectroscopy. The binding affinity of the alkylated peptides with Bcl-x(L) was determined in their dark-adapted and irradiated states by fluorescence anisotropy measurements, and use of different cysteine spacings allowed either activation or deactivation of the binding activities of these peptide-based switches by application of light pulses. Helix-stabilized peptides exhibited high Bcl-x(L) binding affinity with dissociation constants of 42+/-9, 21+/-1, and 55+/-4 nM for Bak(i+ 7)(72-87), Bak i+ 11)(72-87), and Bid(i+ 4)(91-111), respectively (superscript numbers refer to the spacing between cysteine residues), and up to 20-fold enhancements in affinity in relation to their helix-destabilized forms. Bak(i+ 7)(72-87), Bak(i+ 11)(72-87), and Bid(i+ 4)(91-111) each displayed more than 200-fold selectivity for binding to Bcl-x(L) over Hdm2, which is targeted by the N-terminal helix of the tumor suppressor p53. Structural studies by NMR spectroscopy demonstrated that the peptides bind to the same cleft in Bcl-x(L) as the wild-type peptide regardless of their structure. This work opens the possibility of using such photocontrollable peptide-based switches to interfere reversibly and specifically with biomacromolecular interactions to study and modulate cellular function.
authors.affiliation: School of Chemistry, Cardiff University, Park Place, Cardiff, UK.
authors.forename: Sabine
authors.initials: S
authors.lastname: Kneissl
authors.forename: E Joel
authors.initials: EJ
authors.lastname: Loveridge
authors.forename: Christopher
authors.initials: C
authors.lastname: Williams
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Crump
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D002942:Circular Dichroism; D005454:Fluorescence Polarization; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects*; D010777:Photochemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet; D051020:bcl-X Protein / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010455:Peptides; D051020:bcl-X Protein