Design of amphiphilic protein maquettes: enhancing maquette functionality through binding of extremely hydrophobic cofactors to lipophilic domains.
We demonstrate coordination of the extremely hydrophobic 13(2)-OH-Ni-bacteriochlorophyll (Ni-BChl) to the lipophilic domain of a novel, designed amphiphilic protein maquette (AP3) dispersed in detergent micelles [Discher et al. (2005) Biochemistry 44, 12329-12343]. Sedimentation velocity and equilibrium experiments and steady-state absorption spectra indicate that Ni-BChl-AP3 is a four-helix bundle containing one Ni-BChl axially ligated by one or two histidines. The nature of the ligation was pursued with ultrafast visible spectroscopy. While it is well established that light excitation of axially ligated mono- and bisimidazole Ni-BChl in solution leads to rapid imidazole dissociation and nanosecond recombination, there is no evidence of axial ligand dissociation in the light-excited Ni-BChl-AP3. This indicates that Ni-BChl is confined within the AP3 protein, ligated to histidines with severely restricted mobility. Dissociation constants show that Ni-BChl binding to AP3 is considerably weaker than the nanomolar range usual for heme and hydrophilic (HP) maquettes; moreover, there is a tendency for the Ni-BChl-AP3 four-helix bundles to dimerize into eight-helix bundles. Nevertheless, the preparation of the Ni-BChl-AP3 four-alpha-helix maquettes, supported by time-resolved spectroscopic analysis of the nature of the ligation, provides a viable new approach to AP maquette designs that address the challenges involved in binding extremely hydrophobic cofactors.
Noy
Dror
D
Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Discher
Bohdana M
BM
Rubtsov
Igor V
IV
Hochstrasser
Robin M
RM
Dutton
P Leslie
PL
D001665:Binding Sites; D007700:Kinetics; D050356:Lipid Metabolism; D011487:Protein Conformation; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
D011506:Proteins