ja307426k_si_001.pdf (1005.04 kB)
Origin of the Conformational Heterogeneity of Cardiolipin-Bound Cytochrome c
journal contribution
posted on 2016-02-20, 06:14 authored by Yuning Hong, Julia Muenzner, Sebastian
K. Grimm, Ekaterina V. PletnevaInteractions of cytochrome c (cyt c) with cardiolipin (CL) partially unfold the protein, activating
its peroxidase function, a critical event in the execution of apoptosis.
However, structural features of the altered protein species in the
heterogeneous ensemble are difficult to probe with ensemble averaging.
Analyses of the dye-to-heme distance distributions P(r) from time-resolved FRET (TR-FRET) have uncovered
two distinct types of CL-bound cyt c conformations,
extended and compact. We have combined TR-FRET, fluorescence correlation
spectroscopy (FCS), and biolayer interferometry to develop a systematic
understanding of the functional partitioning between
the two conformations. The two subpopulations are in equilibrium with
each other, with a submillisecond rate of conformational exchange
reflecting the protein folding into a compact non-native state, as
well as protein interactions with the lipid surface. Electrostatic
interactions with the negatively charged lipid surface that correlate
with physiologically relevant changes in CL concentrations strongly
affect the kinetics of cyt c binding and conformational
exchange. A predominantly peripheral binding mechanism, rather than
deep protein insertion into the membrane, provides a rationale for
the general denaturing effect of the CL surface and the large-scale
protein unfolding. These findings closely relate to cyt c folding dynamics and suggest a general strategy for extending the
time window in monitoring the kinetics of folding.