Publications


Single Molecule Studies of E-ColiF(1)F(o) ATP Synthase in Lipid Bilayers

BIOPHYSICAL JOURNAL 98 (2010) 54A-54A

WM Ho, RM Berry


An introduction to the physics of the bacterial flagellar motor: A nanoscale rotary electric motor

Contemporary Physics 50 (2009) 617-632

MAB Baker, RM Berry

Biological molecular motors show us how directed motion can be generated by nanometre-scale devices that work at the energy scale of the thermal bath. Direct and indirect observations of functioning single molecule motors allow us to see fundamental processes of statistical physics unfolding in microscopic detail at room temperature, something that was unimaginable only a few decades ago. In this review, we introduce molecular motors and the physics relevant to their mechanisms before focusing on our recent experiments on the bacterial flagellar motor, the rotary device responsible for bacterial locomotion.


Model studies of the dynamics of bacterial flagellar motors.

Biophys J 96 (2009) 3154-3167

F Bai, C-J Lo, RM Berry, J Xing

The bacterial flagellar motor is a rotary molecular machine that rotates the helical filaments that propel swimming bacteria. Extensive experimental and theoretical studies exist on the structure, assembly, energy input, power generation, and switching mechanism of the motor. In a previous article, we explained the general physics underneath the observed torque-speed curves with a simple two-state Fokker-Planck model. Here, we further analyze that model, showing that 1), the model predicts that the two components of the ion motive force can affect the motor dynamics differently, in agreement with latest experiments; 2), with explicit consideration of the stator spring, the model also explains the lack of dependence of the zero-load speed on stator number in the proton motor, as recently observed; and 3), the model reproduces the stepping behavior of the motor even with the existence of the stator springs and predicts the dwell-time distribution. The predicted stepping behavior of motors with two stators is discussed, and we suggest future experimental procedures for verification.


A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor.

Proc Natl Acad Sci U S A 106 (2009) 11582-11587

T Pilizota, MT Brown, MC Leake, RW Branch, RM Berry, JP Armitage

Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stop-start flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides--using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27-28 discrete angles, locked in place by a relatively high torque, approximately 2-3 times its stall torque.


A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

Proceedings of the National Academy of Sciences of the United States of America 106 (2009) 11582-11587

T Pilizota, MT Brown, MC Leake, RW Branch, RM Berry, JP Armitage

Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stopstart flagellar motor. Here,weasked,howdoes the binding of CheY-P stop the motor in R. sphaeroides - using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27-28 discrete angles, locked in place by a relatively high torque, approximately 2-3 times its stall torque.


Characterization and application of controllable local chemical changes produced by reagent delivery from a nanopipet.

Journal of the American Chemical Society 130 (2008) 10386-10393

JD Piper, C Li, C-J Lo, R Berry, Y Korchev, L Ying, D Klenerman

We introduce a versatile method that allows local and repeatable delivery (or depletion) of any water-soluble reagent from a nanopipet in ionic solution to make localized controlled changes in reagent concentration at a surface. In this work, Na(+) or OH(-) ions were dosed from the pipet using pulsed voltage-driven delivery. Total internal reflection fluorescence from CoroNa Green dye in the bath for Na(+) ions or fluorescein in the bath for pH quantified the resulting changes in local surface concentration. These changes had a time response as short as 10 ms and a radius of 1-30 microm and depended on the diameter of the pipet used, the applied voltage, and the pipet-surface separation. After the pipet dosing was characterized in detail, two proof-of-concept experiments on single cells and single molecules were then performed. We demonstrated local control of the sodium-sensitive flagellar motor in single Escherichia coli chimera on the time scale of 1 s by dosing sodium and monitoring the rotation of a 1 microm diameter bead fixed to the flagellum. We also demonstrated triggered single-molecule unfolding by dosing acid from the pipet to locally melt individual molecules of duplex DNA, as observed using fluorescent resonance energy transfer.


Bacterial flagellar motor.

Q Rev Biophys 41 (2008) 103-132

Y Sowa, RM Berry

The bacterial flagellar motor is a reversible rotary nano-machine, about 45 nm in diameter, embedded in the bacterial cell envelope. It is powered by the flux of H+ or Na+ ions across the cytoplasmic membrane driven by an electrochemical gradient, the proton-motive force or the sodium-motive force. Each motor rotates a helical filament at several hundreds of revolutions per second (hertz). In many species, the motor switches direction stochastically, with the switching rates controlled by a network of sensory and signalling proteins. The bacterial flagellar motor was confirmed as a rotary motor in the early 1970s, the first direct observation of the function of a single molecular motor. However, because of the large size and complexity of the motor, much remains to be discovered, in particular, the structural details of the torque-generating mechanism. This review outlines what has been learned about the structure and function of the motor using a combination of genetics, single-molecule and biophysical techniques, with a focus on recent results and single-molecule techniques.


Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli.

J Mol Biol 376 (2008) 1251-1259

Y Inoue, C-J Lo, H Fukuoka, H Takahashi, Y Sowa, T Pilizota, GH Wadhams, M Homma, RM Berry, A Ishijima

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na(+)-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na(+)). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at approximately 2200 pN nm) from stall up to a "knee" speed of approximately 420 Hz, and then falls linearly with speed, extrapolating to zero torque at approximately 910 Hz. Motors containing one to five stators generated approximately 200 pN nm per stator at speeds up to approximately 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to approximately 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to approximately 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.


Characterization and Application of Controllable Local Chemical Changes Produced by Reagent Delivery from a Nanopipet.

J. Am. Chem. Soc. 130 (2008) 10387-10393

JD Piper, C Li, C-J Lo, R Berry, Y Korchev, L Ying, D Klenerman


Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.

Proc Natl Acad Sci U S A 105 (2008) 15376-15381

MC Leake, NP Greene, RM Godun, T Granjon, G Buchanan, S Chen, RM Berry, T Palmer, BC Berks

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of approximately 25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains approximately 15 mobile TatA complexes and a pool of approximately 100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.


Microbiology. How bacteria change gear.

Science 320 (2008) 1599-1600

RM Berry, JP Armitage


How bacteria change gear

Science 320 (2008) 1599-1600

RM Berry, JP Armitage

Bacterial motility is arrested when a protein that acts as a clutch disables rotation of the flagellar motor.


A programmable optical angle clamp for rotary molecular motors.

Biophys J 93 (2007) 264-275

T Pilizota, T Bilyard, F Bai, M Futai, H Hosokawa, RM Berry

Optical tweezers are widely used for experimental investigation of linear molecular motors. The rates and force dependence of steps in the mechanochemical cycle of linear motors have been probed giving detailed insight into motor mechanisms. With similar goals in mind for rotary molecular motors we present here an optical trapping system designed as an angle clamp to study the bacterial flagellar motor and F(1)-ATPase. The trap position was controlled by a digital signal processing board and a host computer via acousto-optic deflectors, the motor position via a three-dimensional piezoelectric stage and the motor angle using a pair of polystyrene beads as a handle for the optical trap. Bead-pair angles were detected using back focal plane interferometry with a resolution of up to 1 degrees , and controlled using a feedback algorithm with a precision of up to 2 degrees and a bandwidth of up to 1.6 kHz. Details of the optical trap, algorithm, and alignment procedures are given. Preliminary data showing angular control of F(1)-ATPase and angular and speed control of the bacterial flagellar motor are presented.


Single-molecule fluorescence microscopy of the twin-arginine translocation (Tat) system

BIOPHYS J (2007) 527A-527A

MC Leake, NP Greene, RM Godun, T Palmer, RM Berry, BC Berks


A programmable optical angle clamp for rotary molecular motors

BIOPHYS J (2007) 372A-372A

T Pilizota, T Bilyard, F Bai, RM Berry


Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load.

Biophys J 93 (2007) 294-302

C-J Lo, MC Leake, T Pilizota, RM Berry

Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.


Stoichiometry and turnover in single, functioning membrane protein complexes

Nature 443 (2006) 355-358

RM Berry, Armitage JP, Chandler JH, Leake MC


The maximum number of torque-generation units in the flagellar motor of Escherichia coli is at least 11

Proceedings of the National Academy of Sciences of the USA 103 (2006) 8066-8071

RM Berry, Chandler JH, Leake MC, Reid SW


Stoichiometry and turnover in single, functioning membrane protein complexes

Nature 443 (2006) 355-358

MC Leake, JH Chandler, GH Wadhams, F Bai, RM Berry, JP Armitage

Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP-MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains ∼22 copies of GFP-MotB, consistent with ∼11 stators each containing two MotB molecules. We also observed a membrane pool of ∼200 GFP-MotB molecules diffusing at ∼0.008 μm2s-1. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP-MotB between the membrane pool and motor with a rate constant of the order of 0.04 s-1: the dwell time of a given stator in the motor is only ∼0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine. © 2006 Nature Publishing Group.


The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11

Proceedings of the National Academy of Sciences of the United States of America 103 (2006) 8066-8071

SW Reid, MC Leake, JH Chandler, CJ Lo, JP Armitage, RM Berry

Torque is generated in the rotary motor at the base of the bacterial flagellum by ion translocating stator units anchored to the peptidoglycan cell wall. Stator units are composed of the proteins MotA and MotB in proton-driven motors, and they are composed of PomA and PomB in sodium-driven motors. Strains of Escherichia coli lacking functional stator proteins produce flagella that do not rotate, and induced expression of the missing proteins leads to restoration of motor rotation in discrete speed increments, a process known as "resurrection." Early work suggested a maximum of eight units. More recent indications that WT motors may contain more than eight units, based on recovery of disrupted motors, are inconclusive. Here we demonstrate conclusively that the maximum number of units in a motor is at least 11. Using back-focal-plane interferometry of 1-μm polystyrene beads attached to flagella, we observed at least 11 distinct speed increments during resurrection with three different combinations of stator proteins in E. coli. The average torques generated by a single unit and a fully induced motor were lower than previous estimates. Speed increments at high numbers of units are smaller than those at low numbers, indicating that not all units in a fully induced motor are equivalent. © 2006 by The National Academy of Sciences of the USA.

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