22 research outputs found
A Map of Dielectric Heterogeneity in a Membrane Protein: the Hetero-Oligomeric Cytochrome b 6 f Complex
The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidationâreduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay hemeâheme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidationâreduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structureâfunction parameter of membrane protein complexes
The Colicin E1 TolC-Binding Conformer: Pillar or Pore Function of TolC in Colicin Import?
The
mechanism by which the drug export protein TolC is utilized
for import of the cytotoxin colicin E1 across the outer membrane and
periplasmic space is addressed. Studies of the initial binding of
colicin E1 with TolC, occlusion of membrane-incorporated TolC ion
channels, and the structure underlying the colicinâTolC complex
were based on the interactions with TolC of individual colicin translocation
domain (T-domain) peptides from a set of 19 that span different segments
of the T-domain. These studies led to identification of a short 20-residue
segment 101â120, a âTolC boxâ, located near the
center of the colicin T-domain, which is necessary for binding of
colicin to TolC. Omission of this segment eliminated the ability of
the T-domain to occlude TolC channels and to co-elute with TolC on
a size-exclusion column. Far-ultraviolet circular dichroism spectral
and thermal stability analysis of the structure of T-domain peptides
implies (i) a helical hairpin conformation of the T-domain, (ii) the
overlap of the TolC-binding site with a hinge of the helical hairpin,
and (iii) a TolC-dependent stage of colicin import in which a central
segment of the T-domain in a helical hairpin conformation binds to
the TolC entry port following initial binding to the BtuB receptor.
These studies provide the first structure-based information about
the interaction of colicin E1 with the unique TolC protein. The model
inferred for binding of the T-domain to TolC implies reservations
about the traditional model for colicin import in which TolC functions
to provide a channel for translocation of the colicin in an unfolded
state across the bacterial outer membrane and a large part of the
periplasmic space