Name: use of stopped-flow fluorescence and labeled nucleotides to analyze the atp turnover cycle of kinesins
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Description: Watch this Scientific Journal Video about Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins at JoVE.com

This work is supported by the Biotechnology and Biological Sciences Research Council (BBSRC), the Royal Society and the University of Nottingham.

1. Determination of the Fluorescence Intensity Change upon Binding of Mant-labeled Nucleotide.
<ol>
	<li>Add 1 &#956;M mant-labeled nucleotide to 1 &#956;M kinesin (final concentrations) in an appropriate reaction buffer. Typically a buffer that benefits microtubule growth and stability is used (see discussion). A commonly used buffer contains 80mM piperazine-bis-2-ethanesulfonic acid (PIPES) pH6.9, 1mM MgCl2 and 1mM ethylene glycol-bis-2 aminoether-tetraacetic acid (EGTA). 
	Note: The concentration of kinesin stated in all protocols refers to the concentration of nucleotide binding sites (i.e., motor domains). Different kinesins exist as monomers, dimers or tetramers and so can contain more than one nucleotide-binding site per molecule.</li>
	<li>After an incubation period (1-3 min) and using a standard fluorimeter, measure the emission spectrum for the mant-labeled nucleotide-kinesin complex. Use an excitation wavelength of 365nm and measure emitted light from 400-500 nm in 1nm increments.</li>
	<li>Make a solution of 1mM mant-labeled nucleotide in the same reaction buffer used in Step 1.1.</li>
	<li>Measure the emission spectrum for the mant nucleotide only sample, as previously described (see Step 1.2).</li>
	<li>Normalize the spectra to the signal at the wavelength of peak fluorescence intensity of the spectrum of mant nucleotide in solution (i.e., without kinesin) by dividing through by this value.</li>
	<li>Plot the normalized spectra (e.g., Figure 2B) to observe fluorescence intensity differences between the mantATP-bound and/or mantADP-bound kinesin and mant nucleotide in solution.</li>
</ol>
2. Measurement of the Association and Dissociation Rate Constants for mantATP
<ol>
	<li>To a solution of up to 20 &#956;M kinesin (see discussion) in any appropriate Mg2+-free buffer (e.g., 80mM PIPES pH6.9, 1mM EGTA, 0.1% Tween20), add 1 mM (final concentration) ethylenediaminetetraacetic acid (EDTA) and 1 mM (final concentration) dithiothreitol (DTT). &#160; Note: Addition of Tween20 to the buffer is recommended as it reduces/prevents loss of kinesin protein on the column used for buffer exchange (Steps 2.3 and 3.3)</li>
	<li>Incubate the solution at 25 &#176;C for 15 min.</li>
	<li>Separate free nucleotide and EDTA from the kinesin using a gravity flow G-25 sephadex gel filtration column according to manufacturer&#8217;s instructions (see list of materials).
	<ol>
		<li>Equilibrate the column with at least 3 column volumes of an appropriate Mg2+-free buffer (see Step 2.1). Load the kinesin-containing solution onto the column.&#160;Prepare a collection tube by adding an appropriate volume of 100mM magnesium chloride solution to the empty tube, such that the final concentration of magnesium chloride is 1 mM after collection of the kinesin solution. &#160;Immediate addition of magnesium chloride helps stabilise the nucleotide-free kinesin.</li>
		<li>Elute the kinesin using&#160;an appropriate Mg2+-free buffer. Kinesins are unstable when free of nucleotide so it is important to work quickly. Store the nucleotide-free kinesin on ice and use within 1-2 hr.</li>
	</ol>
	</li>
	<li>During or prior to preparation of nucleotide-free kinesin, prepare a mantATP concentration series in the same reaction buffer as the final solution of nucleotide-free kinesin. Determine the range of concentrations of mantATP required according to the available concentration of kinesin with a typical series ranging from 0.1 to 50 &#956;M (see step 2.5 and discussion).</li>
	<li>Prepare a concentration series of nucleotide-free kinesin. For each concentration of mantATP (Step 2.4), a concentration of nucleotide-free kinesin should be prepared such that the mant-labeled nucleotide is in 5 to 10-fold molar excess over kinesin nucleotide binding sites.</li>
	<li>Set the excitation wavelength on the stopped-flow fluorimeter to 365 nm and collect emitted light at &#62;400 nm using a long-pass filter.</li>
	<li>Starting with the lowest concentration of mantATP, mix the mantATP with an appropriate concentration of nucleotide-free kinesin in a 1:1 v/v ratio using a stopped-flow fluorimeter (Figure 3A and 3B Inset). Note: The reactants are diluted by 50% upon mixing. Keep a note of the nucleotide concentration both pre and post mixing (i.e., 3.0/1.5 &#956;M) to avoid confusion.</li>
	<li>Fit the change in fluorescence intensity measured at each concentration of mantATP to an exponential function plus a line of constant negative slope to account for photobleaching of the mant group (i.e., Fluorescence = A0.exp(kobs.t)+(m.t+c), where A0 is the amplitude, kobs is the rate constant and t is time, see Figure 3B and discussion). From these fits determine a rate constant (kobs) at each concentration of mantATP. Each kobs is a convolution of an association and dissociation rate constant (Figure 1, k1 and k-1).</li>
	<li>Deconvolve the association and dissociation rate constants by plotting kobs against mantATP concentration (e.g., Figure 3C). Fit these data to a linear function (i.e., kobs = k1[mantATP] + k-1) to obtain the gradient and y-axis intercept. The gradient represents the rate constant for mantATP association (Figure 1, k1) with the units M-1s-1. The intercept represents the dissociation rate constant (Figure 1, k-1) with units of s-1 (see discussion). Note: This approach can also be applied to determine the ADP association and dissociation rate constants (Figure 1, k-4 and k4) by using mantADP as the nucleotide.</li>
</ol>
3. Measurement of the Dissociation Rate Constant for mantADP
<ol>
	<li>To a solution of 2 &#956;M kinesin in a suitable reaction buffer (e.g., 80 mM PIPES pH6.9, 1mM MgCl2, 1mM EGTA, 0.1% Tween20), add 50 &#956;M mantADP (final concentration).</li>
	<li>Incubate at 25 oC for 30 min.</li>
	<li>Remove excess mantADP and other free nucleotide by buffer exchanging the kinesin into the chosen reaction buffer using a G-25 sephadex column (see list of materials) according to manufacturer&#8217;s instructions. The kinesin motor domain should now be loaded with mantADP.</li>
	<li>Set the excitation wavelength on the stopped-flow fluorimeter to 365 nm and collect emitted light at &#62;400 nm using a long-pass filter.</li>
	<li>Mix the mantADP.kinesin complex (~1&#956;M) with a 50-fold or greater molar excess of unlabeled ATP in a 1:1 v/v ratio using a stopped-flow fluorimeter (Figure 4A and 4B Inset).</li>
	<li>Fit the decrease in fluorescence intensity observed over time to a single exponential function plus a line of constant negative slope to account for photobleaching of the mant group (i.e., Fluorescence = A0.exp(kobs.t)+(m.t+c), where A0 is the amplitude, kobs is the rate constant and t is time, see Figure 4B and discussion). The kobs determined from this fit is the rate constant for dissociation of ADP (Figure 1, k4) with units of s-1 (see discussion).</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+the+interaction+of+2'(3')-O-(N-methylanthraniloyl)-ATP+with+myosin+subfragment+1+and+actomyosin+subfragment+1:+characterization+of+two+acto-S1-ADP+complexes.">Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes.</a> Biochemistry. 30, 422-430 (1991).</li><li>Hackney, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway+of+ADP-stimulated+ADP+release+and+dissociation+of+tethered+kinesin+from+microtubules.+Implications+for+the+extent+of+processivity.">Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. 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The authors declare that they have no competing financial interests.

Here, we present protocols for observation and analysis of the kinetics of transitions between nucleotide states for a kinesin. These protocols utilize the rapid mixing method stopped-flow in conjunction with fluorescently labeled nucleotides. Methods of this type have been used extensively to study nucleotide binding and dissociation for a variety of kinesins9-11,13-16. The methods described do not allow observation of phosphate release (Figure 1, Step 3). However, stopped-flow fluorescence can be used to observe this transition by using a phosphate sensing protein to couple the production of phosphate to a fluorescence intensity change17. The methods presented use nucleotides labeled with the small fluorophore methylanthraniloyl (mant), which generally exhibit an increase in fluorescence intensity when bound to protein. Furthermore, the mant fluorophore, when conjugated to either the 2&#8217; or 3&#8217; position on the ribose, often has little or no effect on the binding, dissociation or hydrolysis of the nucleotide9-12. Mant-labeled nucleotides are readily available from commercial sources (see list of materials) and are provided as mixtures of the 2&#8217; and 3&#8217; conjugate as they quickly re-equilibrate when purified to a single isomer8. Mant-labeled nucleotides make excellent reporter molecules by which the kinetics of nucleotide binding and dissociation can be observed.
The use of fluorescently labeled nucleotides means that the read out of the assays described is a real time change in fluorescence intensity. This makes these assays ideal for stopped-flow fluorescence, which allows rapid mixing of reagents and real time observation of changes in fluorescence associated with the subsequent reaction. The combination of stopped-flow as the mixing technique and fluorescence as the method of detection produces a system that is relatively sample efficient. For example, to acquire the data shown in Figure 3C requires 0.8&#160;-&#160;1.0 mg of purified kinesin-13, MCAK, and to acquire the data shown in Figure 4B requires ~0.3&#160;mg of purified kinesin-13, MCAK. One drawback of the use of mant as a fluorophore is that it is prone to photobleaching. This can be observed in isolation from kinesin reaction kinetics by mixing a solution of mant-labeled nucleotide with reaction buffer, in the absence of kinesin, in the stopped-flow. A slow decrease in fluorescence intensity is observed as the mant group becomes bleached. Over the range of timescales used in the protocols described photobleaching of the mant group is well described by a linear function. Therefore, a simple way to correct data for photobleaching is to fit to an exponential function with a baseline described by a linear function (i.e., m.t + c) rather than the more common flat baseline, described by a single parameter.
mantATP binding
When measuring rate constants for association and dissociation of mantATP (Section 2) the ADP, which remains associated with the kinesin nucleotide-binding site in the absence of microtubules, must be removed. This allows mantATP association and dissociation (Figure 1, step 1) to be observed in isolation from ADP dissociation. To achieve this the kinesin is incubated with at least a 50-fold excess of EDTA over nucleotide binding sites (Step 2.1). The EDTA sequesters Mg2+ ions causing Mg2+ to dissociate from the nucleotide-binding pocket, which results in release of the nucleotide11. Therefore, when carrying out steps 2.1&#160;-&#160;2.3, it is vital to use a buffer free of Mg2+. The nucleotide-free kinesin protein is buffer exchanged to separate it both from free nucleotide and from EDTA. To accomplish this separation, a disposable gravity-flow gel filtration column is sufficient and is simple and rapid to use (see list of materials). The interaction of mantATP with nucleotide free kinesin is carried out at a range of mantATP concentrations (Step 2.4) to enable deconvolution of the association and dissociation rate constants (Step 2.8). The theory behind experiments of this type is well described in reference 18. In performing these experiments one begins with the lowest concentration of mantATP. This removes the necessity for wash steps between different concentrations. It will likely be necessary to adjust the sensitivity of the stopped-flow fluorimeter as the mantATP concentration is increased.&#160;The mantATP concentration must always be in at least 5-fold excess over the concentration of kinesin nucleotide binding sites to maintain pseudo first order conditions. However, as the concentration of mantATP is increased, the signal change can become swamped as the fraction of nucleotide that binds to kinesin decreases. Therefore, the concentration of the kinesin is also increased for each new concentration of mantATP such that the concentration of mantATP is never greater than 10-fold molar excess over nucleotide binding sites (Step 2.6).
mantADP Dissociation 
Although association and dissociation rate constants for mantADP can be determined by the same method described for mantATP (Section 2). Dissociation of ADP from a kinesin can be directly observed by preloading the nucleotide-binding site with mantADP (Steps 3.1&#8211;3.3) The mantADP.kinesin complex is then mixed with an excess of unlabeled ATP (step 3.5). The excess unlabeled ATP prevents rebinding of mantADP to the kinesin and thereby allows the dissociation reaction (Figure 1, k4) to be observed in isolation from association of mantADP. In this assay the concentration of unlabeled ATP must be at least a 50-fold molar excess of nucleotide binding sites. The theory behind this assay is well described in reference 18. This direct method gives a more accurate value for the dissociation rate constant than the extrapolation to the y-axis described in Step 2.8.
Inclusion of Microtubules
The methods described can also be used to determine the kinetics of nucleotide binding and dissociation in the presence of microtubules. Microtubules are introduced to the nucleotide containing syringe prior to mixing in the stopped-flow:the lower syringe in Figure 3B and 4B (Insets). In this configuration, the kinesin will meet the nucleotide at the same time as it meets the microtubules. Stabilized microtubules are used, where the lifetime of the tubulin polymer is extended by the use of either a stabilizing chemical, such as taxol, or a nonhydrolysable GTP analogue, such as guanosine-5&#8217;-[(&#945;,&#946;)-methyleno]triphosphate (GMPCPP, see list of materials). It is possible to use two cycles of tubulin polymerization with GMPCPP to make microtubules that can be stored for many months in liquid nitrogen9,19. The use of pre-prepared microtubules significantly reduces the practical difficulties in performing these assays. To include microtubules in the assays, it is important to use a reaction buffer suitable for microtubule stability. The buffer of choice is PIPES based, with the most commonly used buffer containing 80&#160;mM PIPES pH 6.9, 1&#160;mM MgCl2 and 1&#160;mM EGTA (known as BRB80)20,21.
The methods described are not restricted to use with kinesins, but can be adapted and applied to any nucleotide binding protein. For example, similar methods have been applied to the ATP turnover cycle of members of the myosin family of molecular motors12,22 and the use of mant labeled guanosine nucleotides further extends methods of this type to GTP hydrolyzing proteins23,24.

The kinesins are a superfamily of proteins characterized by a highly conserved motor domain1. The kinesin motor domain interacts with microtubules in a nucleotide-dependent manner. Members of the kinesin family display a range of functions from translocating kinesins, which carry cargo in a directed fashion along microtubules, to non-translocating microtubule end-binding kinesins, which regulate microtubule dynamics2,3.
Kinesins use the turnover of adensosine-5&#8217;-triphosphate (ATP) to regulate their interaction with the microtubule cytoskeleton4-6. The conformation of the kinesin motor domain and therefore its affinity for the microtubule depends on its nucleotide state: ATP, ADP, ADP.Pi or no nucleotide can be bound (Figure 1). To understand the molecular mechanism of a kinesin, an understanding of the relationship between ATP turnover and microtubule interaction is required. Therefore, a major goal in the kinesin field is to characterize the functional properties of different nucleotide states and the kinetics of the transitions between them both in the presence and absence of microtubules.
<img alt="Figure 1" fo:content-width="2in" width="200" src="/files/ftp_upload/52142/52142fig1highres.jpg" /> 
Figure 1.&#160;The simplest ATP turnover cycle for a kinesin consists of four possible states: nucleotide-free (&#934;), ATP-bound, ADP.Pi-bound and ADP-bound. Each of the four transitions is characterized by a forward and backward rate constant (kx and k-x respectively).
To understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction, one must determine which step in the ATP turnover cycle is rate limiting in the absence of microtubules and then determine how the cycle is altered by the presence of microtubules. The simplest ATP turnover cycle consists of four individual chemicals steps: (1) ATP binding to the empty site (&#934; denotes the empty site, i.e., nucleotide-free kinesin); (2) ATP cleavage; (3) phosphate dissociation and (4) ADP dissociation (Figure 1). The rate-limiting step sets the rate constant for the complete ATP turnover cycle. Therefore, to determine which step is rate limiting each of the individual transitions must be measured and compared to the rate constant for the complete cycle. Methods to measure the overall ATP turnover cycle rate constant are not described here but can be found in reference 7. Here, we describe methods by which rate constants for individual chemical steps can be determined. There are a number of methods available to study individual transitions in the turnover cycle of a nucleotide hydrolyzing protein. The methods presented here take advantage of the properties of nucleotides labeled with the fluorophore methylanthraniloyl, usually referred to as mant, which display a change in fluorescence intensity upon binding to protein8. The mant group is used because its small size means that, when coupled to the ribose (Figure 2A), it generally has little or no effect on the kinetics of nucleotide binding or hydrolysis9-12. Here, we present protocols describing the use of mant-labeled nucleotides, in conjunction with stopped-flow fluorescence, to allow observation of nucleotide binding and dissociation in the ATP turnover cycle of a kinesin.

<table border="0" cellpadding="0" cellspacing="0" width="781">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">Name of Material/Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:136px;">Catalogue Number</td>
			<td style="width:279px;">Comments/Description</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">2&#8217;/3&#8217;-(N-methylanthraniloyl) adenosine-5&#8217;triphosphate&#160;</td>
			<td style="width:147px;">Jena Biosciences</td>
			<td style="width:136px;"># NU-202</td>
			<td style="width:279px;">mantATP</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">2&#8217;/3&#8217;-(N-methylanthraniloyl) adenosine-5&#8217;-diphosphate&#160;</td>
			<td style="width:147px;">Jena Biosciences</td>
			<td style="width:136px;"># NU-201</td>
			<td style="width:279px;">mantADP</td>
		</tr>
		<tr height="134">
			<td height="134" style="height:134px;width:220px;">Mg-ATP</td>
			<td style="width:147px;">Roche</td>
			<td style="width:136px;"># 10519979001</td>
			<td style="width:279px;">The disodium salt of ATP is made to a concentration of 100&#160;mM in 100&#160;mM MgCl2. Concentration should be verified by absorption at 260&#160;nm (e = 15400&#160;M-1cm-1). Solution stored in aliquots at -20&#160;oC.&#160;&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:136px;">BP120-500</td>
			<td style="width:279px;">0.5&#160;M stock solution in water, pH 8.0. HAZARD: powder harmful if inhaled.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:220px;">Dithiothreitol (DTT)</td>
			<td style="width:147px;">Melford</td>
			<td style="width:136px;">MB1015</td>
			<td style="width:279px;">1&#160;M stock solution in water made fresh for use on the same day. HAZARD: powder harmful if inhaled or in contact with the skin.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">NAP-5 column</td>
			<td style="width:147px;">GE Healthcare</td>
			<td style="width:136px;"># 17-0853</td>
			<td style="width:279px;">G-25 sephadex resin gravity-flow gel filtration column</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:220px;">GMPCPP</td>
			<td style="width:147px;">Jena Biosciences</td>
			<td style="width:136px;"># NU-405</td>
			<td style="width:279px;">nonhydrolysable analogue of GTP</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:220px;">Stopped-flow fluorimeter&#160;</td>
			<td style="width:147px;">Applied Photophysics</td>
			<td style="width:136px;">SX20</td>
			<td style="width:279px;">A fluorimeter with some plumbing attached, which allows reagents to be rapidly mixed in the observation cuvette. Thereby allowing the kinetics of reactions on timescales of ~0.1&#160;-&#160;100&#160;s to be monitored.</td>
		</tr>
	</tbody>
</table>

use of stopped-flow fluorescence and labeled nucleotides to analyze the atp turnover cycle of kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

An increase in fluorescence intensity of the mant group is typically observed upon binding of mant-labeled nucleotide to a kinesin. However, the magnitude of such a signal change is dependent on the particular kinesin in question. Therefore, it is useful to perform equilibrium measurements to confirm both the presence and magnitude of a fluorescence intensity change before progressing to kinetic assays. Representative fluorescence spectra for mantATP and mantADP, both in solution and in complex with the kinesin-13, MCAK, are shown in Figure 2B. These data highlight the fluorescence intensity increase displayed by both mantATP and mantADP upon binding to MCAK. The change in fluorescence intensity upon binding of mant nucleotide to a kinesin is used to report on the association and dissociation of both ATP and ADP (Sections 2 and 3). In the case of MCAK, a fluorescence intensity difference is observed between mantATP.kinesin and mantADP.kinesin9.
Figure 3A depicts the reaction that occurs upon mixing mantATP with nucleotide-free kinesin. Representative data illustrating the increase in fluorescence intensity that occurs upon binding of mantATP to the kinesin-13, MCAK is shown in Figure 3B. Data of the type shown in Figure 3B is collected at a range of mantATP concentrations. A plot of the rate constants (kobs) determined versus the mantATP concentration is shown in Figure 3C. Fitting these data to a linear function (i.e., kobs = k1[mantATP] + k-1) allows deconvolution of the rate constants that contribute to kobs (Step 2.8). Therefore, enabling the determination of both the association and the dissociation rate constant for mantATP (Figure 1, k1 and k-1 respectively).
Figure 4A depicts the reaction which occurs upon mixing mantADP.kinesin with an excess of unlabeled ATP. The data in Figure 4B shows the fluorescence intensity decrease observed for mantADP upon dissociation from the kinesin-13, MCAK. By fitting this data to an exponential function (Step 3.6) the rate constant for dissociation of mantADP is determined directly.
<img alt="Figure 2" src="/files/ftp_upload/52142/52142fig2highres.jpg" /> 
Figure 2.&#160;Nucleotides labeled with the fluorophore methylanthraniloyl (mant) are used to isolate individual steps in the ATP turnover cycle. (A) Structure of mantATP showing the position of the mant fluorophore conjugated to either the 2&#8217; or 3&#8217; position on the ribose. (B) Normalized fluorescence spectra for mantATP and mantADP in solution (mAXP) and mantATP and mantADP in complex with the kinesin MCAK (mATP+MCAK and mADP+MCAK). The mantATP spectra are normalized to the fluorescence at max for mantATP in solution and the mantADP spectra are normalized to fluorescence at max for mantADP in solution. The spectra of the mant nucleotides mixed with MCAK are collected at 1&#160;min post mixing. The concentration of reagents and fluorimeter settings are as described in protocol steps 1.1 and 1.2.
<img alt="Figure 3" src="/files/ftp_upload/52142/52142fig3highres.jpg" /> 
Figure 3.&#160;Measurement of the association rate constant of mantATP. (A) Schematic depicting the reaction which takes place post mixing by stopped-flow: mantATP (mATP) binds to nucleotide-free kinesin. The fluorescence intensity of the mant group increases upon binding to the protein. (B) Fluorescence signal change (black) observed upon mixing mantATP (25&#160;M) with MCAK (5&#160;M nucleotide-free, ). This data is fit (red dashed) to a single exponential function plus a line of constant negative slope, which accounts for photobleaching of the mant fluorophore (see discussion). Inset: Contents of the syringes prior to mixing on the stopped-flow fluorimeter. (C) Rate constants (kobs) determined for the reaction of mantATP with nucleotide-free MCAK plotted against concentration of mantATP. This plot will have a linear region with the gradient being the association rate constant (Figure 1, k1) with units of M-1 s-1 and the y-intercept being the dissociation rate constant (Figure 1, k-1) with units of s-1. <a href="https://www.jove.com/files/ftp_upload/52142/52142fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52142/52142fig4highres.jpg" /> 
Figure 4.&#160;Measurement of the dissociation rate constant for mantADP. (A) Schematic depicting the reaction which takes place post mixing by stopped-flow: kinesin preloaded with mantADP is diluted into an excess of unlabeled ATP. Dissociating mantADP is replaced by unlabeled ATP, thereby preventing the back reaction of mantADP association (see discussion). The fluorescence intensity of the mant group decreases upon dissociation from the protein. (B) Fluorescence decrease observed upon the dissociation of mantADP from the kinesin MCAK. The fluorescence signal (black) is fit (red dashed) to a single exponential plus a line of constant negative slope to account for photobleaching of the mant group (see discussion). The rate constant determined is the rate constant for dissociation of mantADP (Figure 1, k4). Inset: Contents of the syringes prior to mixing on a stopped-flow fluorimeter. <a href="https://www.jove.com/files/ftp_upload/52142/52142fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins at JoVE.com

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

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

use-stopped-flow-fluorescence-labeled-nucleotides-to-analyze-atp

Research

JoVE Journal

Chemistry

Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of molecular iodine to samarium metal in THF.1,2-3 It is a mild and selective single electron reductant and its versatility is a result of its ability to initiate a wide range of reductions including C-C bond-forming and cascade or sequential reactions. SmI2 can reduce a variety of functional groups including sulfoxides and sulfones, phosphine oxides, epoxides, alkyl and aryl halides, carbonyls, and conjugated double bonds.2-12 One of the fascinating features of SmI-2-mediated reactions is the ability to manipulate the outcome of reactions through the selective use of cosolvents or additives. In most instances, additives are essential in controlling the rate of reduction and the chemo- or stereoselectivity of reactions.13-14 Additives commonly utilized to fine tune the reactivity of SmI2 can be classified into three major groups: (1) Lewis bases (HMPA, other electron-donor ligands, chelating ethers, etc.), (2) proton sources (alcohols, water etc.), and (3) inorganic additives (Ni(acac)2, FeCl3, etc).3
Understanding the mechanism of SmI2 reactions and the role of the additives enables utilization of the full potential of the reagent in organic synthesis. The Sm-Barbier reaction is chosen to illustrate the synthetic importance and mechanistic role of two common additives: HMPA and Ni(II) in this reaction. The Sm-Barbier reaction is similar to the traditional Grignard reaction with the only difference being that the alkyl halide, carbonyl, and Sm reductant are mixed simultaneously in one pot.1,15 Examples of Sm-mediated Barbier reactions with a range of coupling partners have been reported,1,3,7,10,12 and have been utilized in key steps of the synthesis of large natural products.16,17 Previous studies on the effect of additives on SmI2 reactions have shown that HMPA enhances the reduction potential of SmI2 by coordinating to the samarium metal center, producing a more powerful,13-14,18 sterically encumbered reductant19-21 and in some cases playing an integral role in post electron-transfer steps facilitating subsequent bond-forming events.22 In the Sm-Barbier reaction, HMPA has been shown to additionally activate the alkyl halide by forming a complex in a pre-equilibrium step.23
Ni(II) salts are a catalytic additive used frequently in Sm-mediated transformations.24-27 Though critical for success, the mechanistic role of Ni(II) was not known in these reactions. Recently it has been shown that SmI2 reduces Ni(II) to Ni(0), and the reaction is then carried out through organometallic Ni(0) chemistry.28
These mechanistic studies highlight that although the same Barbier product is obtained, the use of different additives in the SmI2 reaction drastically alters the mechanistic pathway of the reaction. The protocol for running these SmI2-initiated reactions is described.

Preparation and Use of Samarium Diiodide SmI2 in Organic Synthesis: The Mechanistic Role of HMPA and NiII Salts in the Samarium Barbier Reaction

A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-Barbier reaction.

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of ...

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...