scholarly journals A multi-state dynamical process underpins mechano-adaptation in the bacterial flagellar motor

2021 ◽  
Author(s):  
Navish Wadhwa ◽  
Alberto Sassi ◽  
Howard C. Berg ◽  
Yuhai Tu
Keyword(s):  
Minimal Model ◽  
Bound States ◽  
Stochastic Dynamics ◽  
Dynamical Process ◽  
Transition Rates ◽  
Flagellar Motor ◽  
Multiple Features ◽  
Binding Rate ◽  
Bacterial Flagellar Motor ◽  
The Individual

Adaptation is a defining feature of living systems. The bacterial flagellar motor adapts to changes in external mechanical environment by adding or removing torque-generating stator units. However, the molecular mechanism for mechanosensitive motor remodeling remains unclear. Here, we induced stator disassembly using electrorotation, followed by the time-dependent assembly of the individual stator units into the motor. From these experiments, we extracted detailed statistics of the dwell times underlying the stochastic dynamics of stator unit binding and unbinding. The dwell time distribution contains multiple timescales, indicating the existence of multiple stator unit states. Based on these results, we propose a minimal model with four stator unit states – two bound states with different unbinding rates, a diffusive unbound state, and a recently described transiently detached state. Our minimal model quantitatively explains multiple features of the experimental data and allows us to determine the transition rates between all four states. Our experiments and modeling point towards an emergent picture for mechano-adaptive remodeling of the bacterial flagellar motor in which torque generated by bound stator units controls their effective unbinding rate by modulating the transition between the two bound states. Furthermore, the binding rate of stator units with the motor has a non-monotonic dependence on the number of bound units, likely due to two counter-acting effects of motor’s rotation on the binding process.

scholarly journals Torque-dependent remodeling of the bacterial flagellar motor

2019 ◽  
pp. 201904577 ◽  
Author(s):  
Navish Wadhwa ◽  
Rob Phillips ◽  
Howard C. Berg
Keyword(s):  
Free Energy ◽  
Self Assembly ◽  
Scientific Literature ◽  
Protein Complexes ◽  
Flagellar Motor ◽  
Motor Speed ◽  
Binding Rate ◽  
Motor Load ◽  
Bacterial Flagellar Motor

Multisubunit protein complexes are ubiquitous in biology and perform a plethora of essential functions. Most of the scientific literature treats such assemblies as static: their function is assumed to be independent of their manner of assembly, and their structure is assumed to remain intact until they are degraded. Recent observations of the bacterial flagellar motor, among others, bring these notions into question. The torque-generating stator units of the motor assemble and disassemble in response to changes in load. Here, we used electrorotation to drive tethered cells forward, which decreases motor load, and measured the resulting stator dynamics. No disassembly occurred while the torque remained high, but all of the stator units were released when the motor was spun near the zero-torque speed. When the electrorotation was turned off, so that the load was again high, stator units were recruited, increasing motor speed in a stepwise fashion. A model in which speed affects the binding rate and torque affects the free energy of bound stator units captures the observed torque-dependent stator assembly dynamics, providing a quantitative framework for the environmentally regulated self-assembly of a major macromolecular machine.

DIRECTED MOTION OF BROWNIAN MOTORS: A RATCHET MODEL OF TWO-COUPLED MOLECULAR MOTORS

2007 ◽  
Vol 21 (28) ◽  
pp. 1915-1921 ◽  
Author(s):  
SHUTANG WEN ◽  
HONGWEI ZHANG ◽  
LEIAN LIU ◽  
XIAOFENG SUN ◽  
YUXIAO LI
Keyword(s):  
Molecular Motors ◽  
Particle System ◽  
Langevin Equations ◽  
Transition Rates ◽  
Neck Length ◽  
Directed Motion ◽  
Mean Velocity ◽  
Positive Direction ◽  
Brownian Motors ◽  
The Individual

We investigated the motion of two-head Brownian motors by introducing a model in which the two heads coupled through an elastic spring is subjected to a stochastic flashing potential. The ratchet potential felt by the individual head is anti-correlated. The mean velocity was calculated based on Langevin equations. It turns out that we can obtain a unidirectional current. The current is sensitive to the transition rates and neck length and other parameters. The coupling of transition rate and neck length leads to variations both in the values and directions of currency. With a larger neck length, the bi-particle system has a larger velocity in one direction, while with a smaller neck length, it has a smaller velocity in the other direction. This is very likely the case of myosins with a larger neck length and larger velocity in the positive direction of filaments and kinesins with a smaller neck length and smaller velocity in the negative direction of microtubules. We also further investigated how current reversal depended on the neck length and the transition rates.

scholarly journals Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Physical Interaction ◽  
Flagellar Motor ◽  
Intact Cells ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cross Links ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

scholarly journals Impact of fluorescent protein fusions on the bacterial flagellar motor

2017 ◽  
Author(s):  
M Heo ◽  
AL Nord ◽  
D Chamousset ◽  
E van Rijn ◽  
HJE Beaumont ◽  
...  
Keyword(s):  
Side Effects ◽  
Protein Function ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Switching Frequency ◽  
Flagellar Motor ◽  
Internal Dynamics ◽  
Biologically Relevant ◽  
Bacterial Flagellar Motor

AbstractFluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects. Such insight, however, is still lacking for many applications of fluorescent fusions. This is particularly relevant in the study of the internal dynamics of motor protein complexes, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to thestatorof the bacterial flagellar motor (BFM) complex have previously been used to successfully unveil the internal subunit dynamics of the motor. Here we report the effects of three different fluorescent proteins fused to the stator, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry in the complex remained unaffected. MotB fusions decreased the rotation-direction switching frequency of single motors and induced a novel BFM behavior: a bias-dependent asymmetry in the speed attained in the two rotation directions. All these effects could be mitigated by the insertion of a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions on BFM dynamics and their alleviation—new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Author summaryMuch of what is known about the biology of proteins was discovered by fusing them to fluorescent proteins that allow detection of their location. But the label comes at a cost: the presence of the tag can alter the behavior of the protein of interest in unforeseen, yet biologically relevant ways. These side effects limit the depth to which fluorescent proteins can be used to probe protein function. One of the systems that has been successfully studied with fluorescent fusions for which these effects have not been addressed are dynamic protein complexes that carry out mechanical work. We examined how fluorescent proteins fused to a component of the bacterial flagellar motor complex impacts its function. Our findings show that the fusion proteins altered biologically relevant dynamical properties of the motor, including induction of a novel mechanical behavior, and demonstrate an approach to alleviate this. These results advance our ability to dissect the bacterial flagellar motor, and the internal dynamics of protein complexes in general, with fluorescent fusion proteins while causing minimal perturbation.

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