TY - JOUR
T1 - Impact of distant charge reversals within a robust β-barrel protein pore
AU - Mohammad, Mohammad M.
AU - Movileanu, Liviu
N1 - Copyright:
Copyright 2015 Elsevier B.V., All rights reserved.
PY - 2010/7/8
Y1 - 2010/7/8
N2 - Among all β-barrel pores, staphylococcal aα-hemolysin (aαHL), a heptameric transmembrane protein of known high-resolution crystal structure, features a high stability in planar lipid bilayers under a wide range of harsh experimental conditions. Here, we employed single-channel electrical recordings and standard protein engineering to explore the impact of two distant charge reversals within the interior of the β-barrel part of the pore. The charge reversals were replacements of lysines with aspartic acids. A charge reversal within the structurally stiff region of the β barrel near the pore constriction reduced the open-state current of the pore, but produced a quiet pore, showing current fluctuation-free channel behavior. In contrast, a charge reversal on the trans entrance, within the structurally flexible glycine-rich turn of the β barrel, increased the open-state current and produced gating activity of the pore in the form of large-amplitude and frequent current fluctuations. Remarkably, cumulative insertion of the two distant charge reversals resulted in a large-amplitude permanent blockade of the β barrel, as judged by both single-channel and macroscopic current measurements. The results from this work suggest that these distant charge reversals are energetically coupled, producing different impacts on the ionic transport, the unitary conductance and the open-state probability of the pore.
AB - Among all β-barrel pores, staphylococcal aα-hemolysin (aαHL), a heptameric transmembrane protein of known high-resolution crystal structure, features a high stability in planar lipid bilayers under a wide range of harsh experimental conditions. Here, we employed single-channel electrical recordings and standard protein engineering to explore the impact of two distant charge reversals within the interior of the β-barrel part of the pore. The charge reversals were replacements of lysines with aspartic acids. A charge reversal within the structurally stiff region of the β barrel near the pore constriction reduced the open-state current of the pore, but produced a quiet pore, showing current fluctuation-free channel behavior. In contrast, a charge reversal on the trans entrance, within the structurally flexible glycine-rich turn of the β barrel, increased the open-state current and produced gating activity of the pore in the form of large-amplitude and frequent current fluctuations. Remarkably, cumulative insertion of the two distant charge reversals resulted in a large-amplitude permanent blockade of the β barrel, as judged by both single-channel and macroscopic current measurements. The results from this work suggest that these distant charge reversals are energetically coupled, producing different impacts on the ionic transport, the unitary conductance and the open-state probability of the pore.
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U2 - 10.1021/jp101311s
DO - 10.1021/jp101311s
M3 - Article
C2 - 20540583
AN - SCOPUS:77954340928
VL - 114
SP - 8750
EP - 8759
JO - Journal of Physical Chemistry B
JF - Journal of Physical Chemistry B
SN - 1520-6106
IS - 26
ER -