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Projects
-Fluorescent flagella
-Swimming without flagella
-Gliding motility
-Twitching motility
-Fluorescent chemotaxis proteins
-Chemotactic signaling studied by FRET
-Motor force generation
-Switching under load
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Chemotactic signaling studied by FRET
In a model bacterium, Escherichia coli, the chemotaxis
pathway is well characterized (Figure
1). Receptors at the cell surface detect changes in the
concentrations of attractants and generate shifts in the level of
phosphorylation of a diffusible signaling protein CheY. Phosphorylated
CheY modulates the direction of flagellar motor rotation and thus
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Figure 1: Signal transduction pathway in chemotaxis of E. coli. Pathway is schematically divided in two parts, excitation and adaptation.
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affects swimming behavior of the cell. The entire signal transduction
pathway includes five attractant-specific receptors (Tsr, Tar, Trg,
Tap, and Aer), six cytoplasmic chemotaxis proteins (CheA, CheW, CheR,
CheB, CheY, and CheZ), and three proteins comprising a switch complex
at the cytoplasmic face of the flagellar motor (FliM, FliN, and FliG).
Based on genetic and biochemical data, functions have been assigned to
all the chemotaxis proteins: CheA is a kinase that phosphorylates the
response regulator CheY and also the methylesterase CheB; CheW is an
adaptor protein, coupling CheA to receptors; CheR, a
methyltransferase, and CheB, a methylesterase, mediate adaptation to a
constant attractant concentration by adjusting the methylation level
of receptors; CheZ is a phosphatase of CheY~P. Despite its relative
simplicity, the chemotaxis system exhibits features common to many
cellular networks, such as signal amplification, integration, and adaptation. This has inspired a
number of recent attempts to mathematically model this pathway.
However, no model has been able to explain the complexity of the
behavioral response. Thus, we need to understand better how the
pathway works in vivo.
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Figure 2: Using FRET between CFP and YFP to measure protein interactions. When CheZ-CFP and CheY-YFP fusion proteins do not interact, illumination with cyan (~430 nm) light results in stronger CFP fluorescence (left). As a result of phosphorylation-induced interactions between CheZ and CheY, CFP and YFP are brought into proximity and excitation energy is transferred, resulting in stronger YFP fluorescence (right).
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To address this question we used a technique called fluorescence resonance energy transfer (FRET).
FRET, which depends on the distance-dependent transfer of energy from
an excited donor fluorophore to an acceptor fluorophore, is one of the
few tools available to study protein interactions in the cell in real
time. In vivo-FRET was greatly advanced by the recent (and
ongoing) improvement in spectral characteristics of green fluorescent
protein (GFP) that led to creation of spectrally shifted variants of
GFP, cyan and yellow fluorescent proteins (CFP and YFP). For Example, genetically
fusing CFP and YFP to CheZ and CheY, respectively, allows one to test their
interactions in vivo (Figure 2).
When cells have adapted, the rate of synthesis of CheY~P, the molecule that couples the receptors to the flagella, is equal to its rate of destruction. The rate of synthesis is proportional to the activity of the receptor kinase, CheA. The rate of destruction is proportional to the concentration of the enzyme substrate complex, CheZ CheY~P, which can be monitored by FRET. So we measure the cyan and yellow fluorescence, add or remove attractant or repellent, and measure the cyan and yellow fluorescence again. From these measurements, we infer the corresponding change in the activity of the receptor kinase.
We found, to our delight, that the fractional change in the activity of the kinase is many times larger than the fractional change in receptor occupancy. That is, there is a large amplification step at the beginning of the sensory transduction pathway. The amplification appears to be due to receptor-receptor interactions. In addition, using FRET between CFP fused to FliM and YFP fused CheY, we have studied the binding of CheY~P to FliM, the interaction that promotes clockwise flagellar rotation.
References
Sourjik, V., and Berg, H.C. “Receptor sensitivity in bacterial chemotaxis.” Proc. Natl. Acad. Sci. USA 99, 123-127 (2002).
Sourjik, V. and Berg, H.C. “Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer.” Proc. Natl. Acad. Sci. USA 99, 12669-12674 (2002).
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