POSTERS - BLAST X - University of Utah

BLAST X Tue. Morning Session
EXPERIMENTAL EVIDENCE FOR CONFORMATIONAL SPREAD IN THE BACTERIAL
SWITCH COMPLEX
Richard W. Branch 1 , Fan Bai 1 , Dan V. Nicolau 2 , Teuta Pilizota 1 , Bradley Steel 1 , Philip K.
Maini 2 , Richard M. Berry 1
1 Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1
3PU, UK. 2 Centre for Mathematical Biology, Mathematical Institute, University of Oxford,
St.Giles Road, Oxford OX1 3LB, UK.
The bacterial switch complex in E. coli controls the direction of rotation of the bacterial
flagellar motor between clockwise and counterclockwise modes. The complex takes the form of
a ring composed of about 110 FliN, 34 FliM and 26 FliG protein subunits. Regulation is through
binding of the signaling molecule CheY-P to FliM. FliG interfaces with the torque-generating
stator units of the motor. The precise mechanism by which the complex executes a switch is
unclear.
The complex displays the ultrasensitive nature typical of allosteric proteins, with a steep
sigmoidal relationship existing between [CheY-P] and motor rotational bias. Allosteric regulation
of proteins has classically been understood in terms of the Monod-Wyman-Changeux (MWC) or
Koshland-Nemethy-Filmer (KNF) models. However, it is unrealistic that MWC-type concerted
transitions could be responsible for quaternary conformational changes of such a large complex,
and cooperative binding studies in vitro and in vivo have precluded a KNF-type induced-fit
mechanism.
The MWC and KNF models are recognized as limiting cases of a general allosteric
scheme that has recently been described in a model of conformational spread. The model has
been shown to be capable of reproducing motor switching kinetics. A directly observable
consequence of conformational spread in the switch complex would be the variation of motor
speed associated with the conformational spread of ring subunit state. In particular, the duration
of switch events should be finite and broadly distributed due to the diffusive random walk of
conformational spread, and incomplete switches should be observed due to incomplete growth
and shrinkage of subunit state domains.
We have used high-resolution back-focal-plane interferometry of polystyrene beads
attached to truncated WT E. coli flagella to resolve intermediate states of the motor predicted by
conformational spread, and demonstrate detailed quantitative agreement between our
measurements and conformational spread simulations. Individual switch events are not
instantaneous, but follow a broad distribution of switch times with mean ~ 20 ms. The shortest
switch events are observed to last less than 1ms, while the longest require over 100ms and take
several revolutions to complete. Intervals between switches are exponentially distributed at all
values of bias. Incomplete switches reaching a range of intermediate speeds are observed. The
events are Poisson distributed in time with a bias-dependent frequency.
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