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Single Molecule Microscopy for Proteins In Vitro

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This figure shows a time sequence (1-s intervals) of three individual kinesin-GFP molecules (pseudocolored green) moving processively on an axonemal microtubule (pseudocolored red) imaged by total internal reflection microscopy.

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Looking at single biomolecules at a time is important to understand how they physically perform their function (movement, conformational changes etc.) in a time course. To be able to see single proteins, we specifically label them with organic fluorophores and shine a laser light to observe the emitted fluorescent light under a microscope. The emitted light is collected with a highly sensitive CCD camera for quantitative analysis.

Our fluorescence microscope is equipped with 488 nm Argon Ion, 532 nm NdYag and 633 nm HeNe lasers for the excitation of wide variety of fluorescent probes. All three lasers are combined to follow the same optical path and focused to the back focal plane of the microscope objective (Nikon 100X, 1.49 NA). The beam is then shifted laterally along the focal plan that creates tilted and parallel laser beam emitted from the microscope objective. When the tilt is larger than the critical angle, transmitted light will be total internally reflected back from the glass/water boundary. Total Internal Reflection (TIR) of light creates an evanescent field which penetrates only 100 nm into the flow chamber. TIRF is ideal to study surface immobilized cells and in vitro systems since it results in minimal fluorescent background and hence sensitively detect fluorescent photons. Compared to the scanning confocal setup, TIRF is a wide field illumination technique that readily allows simultaneous observation of many single biomolecules on the surface. Fluorescent photons are filtered and collected by an EMCCD camera (Photometrics, Cascade 512B) which can detect 92% of incoming photons with a minimal electronic noise.


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We use our single molecule TIR scope to detect fast conformational changes and particle tracking experiments. Specifically, by labeling a single protein with a pair of fluorescent dyes whose spectrum overlaps to allow Fluorescent Resonance Energy Transfer (FRET). Closely spaced fluorophores can transfer their energy via dipole-dipole interaction to each other. Excitation of the donor molecule whose emission is in resonance with the acceptor’s absorption yields the emission of an acceptor. The energy transfer depends on the distance (R) between the donor and the acceptor molecule with R6 that makes energy transfer very sensitive to the distance changes between the donor and the acceptor. Labeling of two sites of a protein with a FRET pair yields information on conformational dynamics for the distances between 2-10 nm. We have used this method recently to measure neck linker conformational changes in moving kinesin molecules.

To track the movement of motor proteins with high precision in vitro, we use Fluorescence Imaging with One Nanometer Accuracy (FIONA). The image of a point-like fluorescent object is as wide as 250 nm in the visible region of the light because of the diffraction limit. The position of an object, however, can be localized very precisely by determining the center of its emission pattern. The precision of measurement depends on collecting thousands of photons per image and minimizing the noise factors. By using optimized oxygen-scavenging and emission stabilizer conditions, millions of photons can be collected from a single molecule before it photobleaches. We obtain 1-2 nm localization of organic dyes (Cy3, TMR, Cy5) within ~100 msec, which enables us to measure the step size of the motor proteins kinesin and cytoplasmic dynein.

To measure the step size of the kinesin motor, we label the motors with a single organic dye. For specific labelling, we removed all the solvent exposed cysteines and place a single cysteine at different positions. Those cysteines were then labelled with a single Cy3 molecule. Labelled motors were introduced into the flow chamber after immobilizing axonemes on the glass surface. (Axoneme is a microtubule rich structure that is more rigid than microtubules, providing better surface immobilization.) At 150 nM ATP, the spots moved an average of 2 nm/sec. We observed that a single labeled kinesin head takes 17.3 ± 3.3 nm steps, while the tail domain is known to move 8.3 nm per step. This experiment provided direct evidence for a hand-over-hand type of movement for kinesin.

To measure the stepwise movement of yeast cytoplasmic dynein, we have inserted a DHA tag into to C-terminus of the motor, which enables covalent linkage of TMR or biotin. We have labelled dyneins with quantum dots that are ~20 times brighter and more stable than Cy3 and TMR dyes. We have found that dynein tail mostly takes 8 nm steps although longer as well as side and backward steps are observed. The head region, on the other hand, advances most frequently by 16 nm steps, indicating that dynein moves by alternately shuffling its two motor rings between rear and forward positions, analogous to the hand-over-hand motion of kinesin.



Fig 1. Fluorescence Resonance Energy Transfer (FRET)

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  Fig 2. Diffraction Limited Spot: (Left) The theoretical image of a point-like fluorescent in the light microscope is called the Point Spread Function. The width of the major peak is roughly 250 nm in the visible region of light. (Right) Pixelated image of a surface immobilized Cy3 dye.


  Fig 3. Fluorescence Imaging with One Nanometer Accuracy (FIONA). (Left) Fluorescent image of single molecules on the glass surface. (Right) Two-dimensional Gaussian fit determines the center of the diffraction-limited spot which represents the position of the molecule. The spot contains 14,000 photons and its center can be determined with 1.5 nm error.

Fig 4. Tracking the stepwise movement of kinesin with FIONA. Kinesin Head advances with 16 nm steps on average.



Fig 5. Quantum Dots can be tracked with up to 8Å precision.



  Fig 6. FIONA assay on quantum dot labelled yeast cytoplasmic dynein reveals that dynein tail mostly takes 8 nm steps whereas the head region takes 16 nm steps.

Movie 1. Movie of Qdot-labeled dynein.

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updated 4/9/07

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