Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The overall goal of this procedure is to measure the kinetics of nucleotide binding and dissociation during the A TP turnover cycle of a kinesin. This is accomplished by first preparing the kinesin protein, either free of nucleotide or loaded with fluorescently labeled nucleotide. Next, the prepared kinesin is mixed with nucleotide using a stopped flow fluorimeter and the change in fluorescence associated with binding or dissociation of fluorescently labeled nucleotide is observed.

Then the change in fluorescence is fit to an exponential function to obtain a rate constant. Finally, the measured rate constants are devolved and rate constants are attributed to particular transitions of the A TP turnover cycle. Ultimately, the combination of fluorescently labeled nucleotide and stopped flow fluorescence is used to measure the kinetics of nucleotide binding and dissociation.

CIN Demonstrating the procedure will be Jennifer Patel postdoc and Hannah Belch, a grad student from my lab. After determining the fluorescence intensity of kinesin upon binding of man labeled nucleotide as outlined in the text protocol to a solution of up to 20 micromolar kinesin in magnesium free buffer at one millimolar of EDTA and one millimolar of DTT incubate the solution for 15 minutes at 25 degrees Celsius, set up a gravity flow G 25 SX gel filtration column and equilibrate with at least three column volumes of a magnesium free buffer. Then load the essin solution into the column to separate free nucleotides and EDTA from the kinesin.

Repair a collection tube by adding in a volume of 100 millimolar magnesium chloride solution to the empty tube, such that the final concentration of magnesium chloride is one millimolar. After collection of the kinesin solution, using an appropriate magnesium free buffer quickly elute the kinesin. The presence of magnesium chloride in the collection tube helps stabilize the kinesin immediately after Ian from the column store the kinesin on ice and use within one to two hours.

In the meantime, prepare a man a TP concentration series. Choose the range of concentrations according to the available concentration of kinesin. For each concentration of mant a TP prepare a suitable concentration of kinesin such that the man labeled nucleotide is in five to tenfold molar excess over kinesin nucleotide binding sites.

Set the excitation wavelength on the stopped flow fluorimeter the 365 nanometers and the emission wavelength above 400 nanometers with a long pass filter. Next, starting with the lowest concentration of Manta TP, use the stopped flow fluorimeter to mix the manta TP and nucleotide free kinesin in a one-to-one volume to volume ratio. Using the following formula fit the change in fluorescence intensity measure to teach concentration of mant A TP to an exponential function, plus a line of constant negative slope to account for photobleaching of the mant group.

From these fits determine a rate constant at each concentration of mant. A TP then devolve the association and dissociation rates by flossing kops against the concentration of mant. A TP Then fit these data to a linear function to obtain the gradient and y axis intercept.

The gradient represents the rate constant for man a TP association with units per molar per second. The intercept represents the dissociation rate constant with the units per second. To measure the dissociation rate constant ferment a DP to a solution of two micromolar kinesin in a suitable buffer at 50.

Micromolar meant a DP incubator 25 degrees Celsius for 30 minutes. After using a G 25 Sedex column to remove fre mant, A DP from the kinesin, loaded with mant A DP, and setting the stop flow fluter as described earlier in this video, use the stopped flow to mix the mant A DP Kinine complex with a 50 fold or greater molar excess of unlabeled A TP in a one-to-one volume to volume ratio fit. The decrease in fluorescence intensity observed over time to a single exponential function, plus a line of constant negative slope to allow for photobleaching of the group.

The kops determined from this fit is the rate constant for dissociation of mant A DP with units of per second. The magnitude of the signal change upon binding or dissociation of mantley wood nucleotide is dependent on the particular kinesin. Therefore, it is useful to first perform equilibrium measurements to confirm both the presence and magnitude of a fluorescent intensity change.

Fluorescent spectra ferment a TP and meant a DP both in solution and in complex of the kinesin M CAC are shown here. This figure depicts the reaction that occurs upon mixing man a TP with nucleotide free kinesin. The increase in fluorescence intensity that occurs upon binding of man A TP to M CAC is shown here, fitting the rate constant versus the man.

A TP concentration to a linear function as seen here allows deconvolution of the rate constant that contribute to kops and provides the association and dissociation rate stance ferment A TP.This figure shows the fluorescence intensity decrease observed ferment A DP upon dissociation from M CEC as illustrated here. The rate constant for dissociation of mant A DP from Kinesin is determined by fitting this data to an exponential function. Once mastered, these protocols can each be completed in less than three hours.

Though this method can provide insights into kinesin, it can also be applied to other nucleotide binding proteins, such as myosin and G proteins. After watching this video, you should have a good understanding of how to use fluorescently labeled nucleotides and a stop flowmeter to measure the kinetics of nucleotide binding and dissociation for a kinesin.