Two-stimuli manipulation of a biological motor

F1-ATPase is an enzyme acting as a rotary nano-motor. During catalysis subunits of this enzyme complex rotate relative to other parts of the enzyme. Here we demonstrate that the combination of two input stimuli causes stop of motor rotation. Application of either individual stimulus did not significantly influence motor motion. These findings may contribute to the development of logic gates using single biological motor molecules.


Findings
Biological nano-scale motors fulfil a broad range of tasks in living cells. Some motors like myosin, kinesin and dynein move in linear fashion. Other motors perform rotary motion, e.g. the bacterial flagellar motor or the enzyme F 1 -ATPase. F 1 -ATPase hydrolyses ATP into ADP and inorganic phosphate. It is the smallest biological rotary motor known, with a total molecular mass of ~400 kDa and the core subunits α 3 β 3 γ [1 -3]. During enzymatic catalysis subunit γ rotates within the hexagonal α 3 β 3 domain. This rotary movement has been microscopically monitored by attachment of large probes such as fluorescently labelled actin filaments and polymer microspheres to subunit γ [4][5][6][7]. In addition to plain motor observation, also manipulation of motor movement has been reported. Rotation in reverse direction was imposed on F 1 -ATPase using magnetic tweezers [8,9]. Furthermore, rotor movement was successfully modulated by chemical signals, including redox-switching [10,11], builtin Zn-sensitive switches [12], small organic molecules [13][14][15] as well as by temperature control [16,17]. However, these experiments describe the response of F 1 to individual stimuli and do not reveal how simultaneously acting stimuli are processed by the motor.
Here we report manipulation of the F 1 -ATPase motor movement at single molecule level by concerted optical and chemical input stimuli. We combined an optical stimulus (high-intensity illumination) with a chemical stimulus (rhodamine 6G), on the rotary movement of single F 1 molecules.

Preparation of F1-ATPase
The α 3 β 3 γ core complex of F 1 -ATPase originating from Bacillus PS3 was prepared as previously described in [10] and hereinafter referred to as F 1 -ATPase. The enzyme was over-expressed in Escherichia coli strain JM103 uncB-D using the pkkHC5 expression plasmid [10]. This plasmid codes for the α, β, and γ subunits of the thermophilic Bacillus PS3 F1-ATPase, carrying a decahistidine tag at the N terminus of the β subunit and the mutation γSer106→Cys.
Rotation Assay F 1 -ATPase was biotinylated at a single cysteine residue in subunit γ using biotin-(PEAC)5-maleimide (Dojindo, Japan), as described elsewhere [10]. The biotinylated F 1 -ATPase (30 nM) in an assay mixture containing 10 mM 3-(N-Morpholino) propanesulfonic acid (MOPS)/KOH (pH 7.0), 50 mM KCl and 2 mM MgCl 2 (buffer A) was infused into a flow cell, constructed from microscope cover slips as described [4], and incubated for 5 min to allow for immobilization. The flow cell was washed with 100 μl of Buffer A supplemented with 10 mg/ml bovine serum albumin (buffer B). Subsequently, a suspension of streptavidin-coated polystyrene beads (Bangs Laboratories, diameter 510 nm) suspended in Buffer B was infused and incubated for 15 min. Next, 100 μl of reaction buffer (Buffer B supplemented with 2 mM ATP, 4 mM MgCl 2 , 2.5 mM phosphoenolpyruvate, and 0.1 mg/ml pyruvate kinase (Roche Applied Science) in the absence or in the presence 100 μM Rhodamine 6G (Merck) was infused and microscopic observation was started. Rotation of beads was observed under bright field illumination with an inverted fluorescence microscope (TI Eclipse, Nikon) equipped by a Nikon Plan. Apo. 100× (N.A. 1.4) objective. Images were recorded with an Andor iXon DU-897BI EMCCD camera (Andor Technology, Belfast, UK) at 25 Hz frame rate. Image analysis was done using self made tracking routines under Matlab (The MathWorks, Natick, USA) and the open-source image analysis software ImageJ. Bright field illumination was performed by an attenuated 100W Halogen lamp (35 mW/cm/ 2 on the sample).
High illumination intensity of the probe was performed by 110 W Mercury lamp in epi-fluorescence illumination. The excitation wavelength was selected by a 540 ± 10 nm interference filter.

Motor movement in absence of input stimuli
ATP-driven rotation of F 1 -ATPase subunit γ was visualized by attachment of a bead to the γ subunit (Fig. 1a) [7,11], typical time courses of the rotational movement of two molecules F 1 are shown in Fig. 1b. Rotation of both singlebead as well as duplex-beads was unidirectional, continuous and directions were always counter-clockwise when viewed from top (Fig. 1b, [6]). Bead rotation occasionally displayed pauses and subsequently resumed rotation. These pauses have been described previously and may be attributed to transient inhibition of F 1 by Mg-ADP [18,19].

Motor response to concerted chemical and physical input
Next, we determined the motor response to concerted physical and chemical stimuli. Illumination of the samples with light at 540 ± 10 nm for 5-10 sec at maximum Rotary movement of F 1 -ATPase motor  intensity (110 W/cm 2 ) in the presence of rhodamine 6G lead to a complete arrest of motor movement within the duration of the light pulse (Fig. 2a). This light-induced motor response was highly reproducible and observed for >90% of all investigated motor molecules (n = 20), with "motor arrest" defined as <1 revolution per minute of a single or a duplex bead. These results indicate that rotation of the F 1 -motor can be stopped by the combination of an optical and a chemical input signal.

Motor response to individual input variables
We have observed a dramatic response of F 1 -ATPase motor movement to two combined inputs. Next, we assessed the two inputs imposed separately on the rotating motor. Firstly we tested the effect of high light intensity on F 1 rotation in the absence of rhodamine 6G. Typically, no significant effect on motor movement was detected (Fig. 2b), only <10% of the observed F 1 -ATPase molecules (n = 22) stopped upon illumination.
We have demonstrated that the movement of a biological motor can be arrested by synergistic inputs of optical and chemical stimuli. Motor arrest is observed at single molecule level and does not occur when the input stimuli are applied separately. The motor response reported here is is consistent with a function as an "AND" logic gate in terms of producing a single output on two concerted inputs [32][33][34]. For full implementation of a motor protein "AND" gate, reversibility of the motor system response is an important factor. Experiments to gain a deeper understanding of the response mechanism and to improve reversibility are on-going in our laboratory. Biomolecules acting as "AND" gates in bulk-phase have been described earlier, e.g. light dependent release of an unfolded fluorescent protein from a chaperone protein [34], or an enzyme-based logic gate [35]. Extending the work of these authors, our results may help to develop motor proteinbased logic gates, operating and monitored at the single molecule level.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
ZR performed motor labelling and microscopic observation, MV prepared the microscope set-up and took images, AD and CG carried out image analysis, KK, WZ HL and DB conceived the experiments, DB coordinated the study. All authors read and approved the final manuscript.
Manipulation of F 1 rotor motion by optical and chemical inputs Figure 2 Manipulation of F 1 rotor motion by optical and chemical inputs. Sequential images of a rotating beads before and after a pulse (10 sec) of high intensity white light illumination (white bar) in the presence (A) or absence (B) of rhodamine 6G. (C) Rotating beads in the presence of rhodamine 6G, but without light pulse.

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