The Ability of Deuterium Oxide to Stabilize the Kinesin Motor Protein

8/4/2019 The Ability of Deuterium Oxide to Stabilize the Kinesin Motor Protein

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THE ABILITY OF DEUTERIUM OXIDE TO STABILIZE THEKINESIN MOTOR PROTEIN

Kenji Doering1, Nadiezda Fernandez-Oropeza*, and Steve Koch*

Department of Electrical and Computer EngineeringUniversity of New Mexico, Albuquerque, NM 87106

Undergraduate student ofDepartment of Mathematics and Department of PhysicsUniversity of WashingtonSeattle, WA 98195

ABSTRACT

Kinesin stability is the key to utilizing this motor protein outside of organisms bodies.Kinesin becomes unstable at temperature of around 45C (Konrad J. Bohm, Roland Stracke,Marina Baum, Martin Zieren, Eberhard Unger 1999). Deuterium oxide is a naturally toxi lic,though it has been experimentally proven to stabilize other proteins and products (Artur Galazka1989, Julie Milstien, Michel Zaffran 2006, Collin S. Pittendrigh, Elizabeth S. Cosbey 1974). Bysuspending the kinesin in deuterium oxide we hope to increase the amount of time the kinesinremains stable for, as well as its thermal stability. Our ovalbumin work shows that the deuteriumoxide stabilizes the protein fairly well, but so far our kinesin experimentation has thus beeninconclusive.

INTRODUCTION

Kinesin is a motor protein within organismsthat uses microtubules to transport otherbiomolecules such as vesicles and other cargo (SeeFigure 1.1). Utilizing ATP through ATP hydrolysisthe kinesins motor domains use a one foot in frontof the other technique, binding and unbinding itsfeet to the microtubules tubulin graduallyprogressing towards its destination, while pullingthe cargo with its cargo binding point. Themicrotubules are made up of the two differentmonomers, alpha and beta tubulin. The direction ofthe kinesin protein movement is determined by the

plus-minus direction orientation of the microtubule.A kinesin molecule moves towards the plus end of amicrotubule only upon the beta tubulin monomers.The ability of kinesin to turn chemical energy(ATP) into mechanical energy (walking) has beenspeculated to have great utility in nanodevices(Moorjani 2003), especially with the knowledge ofhow to control motion direction. Other labs have even determined how to turn the motor

Figure 1.1

A motor protein, kinesin, pulling a vesicle

along a microtubule. Alternating

microtubule colors represent alpha and

beta tubulin.

Image Source: Harvard University

Biovisions

Motor Domains


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domains motions on and off using a Zinc switch through the binding and unbinding of a zincion (George D. Bachand, Adrienne C. Greene, Amanda M. Trent 2008).

The current obstacle with kinesin use is that kinesin is an inherently unstable molecule.With a denaturing temperature at around 45C, practical use of kinesin becomes unrealisticwithout expensive cooling systems. Even when chilled, the kinesin will breakdown and

aggregate over time anyway. Proteins are originally folded into specific patterns that for theprocess they were designed for, so when they are denatured (unfolding) they lose the ability tomaintain that function and are irreversibly damaged (at least in our case for kinesin). Though notnecessarily the cause, kinesin instabilities and mutations have turned up in diseases such asAlzheimers (Stokin 2005) and Amyotropic lateral sclerosis (McMurray 2000), andunderstanding how to prevent these instabilities or slow these instabilities could be critical infuture research

PROJECT

Deuterium oxide, or heavy water, contains the isotope of hydrogen which has both aproton and neutron. By being twice as heavy on the hydrogen side, deuterium oxide becomes aless reactive molecule than regular water. This deuterium oxide molecule has been found to havestabilizing capabilities. Increasing the thermal stability of fruit flies, vaccine stabilizing(ArturGalazka, Julie Milstien, Michel Zaffran 2006, Collin S. Pittendrigh, Elizabeth S. Cosbey 1974),and some have even believed a daily dose of deuterium oxide would increase a personslifetime(Mikhail S. Shchepinov 2007, Fiona Macrae 2008). However, we are not testing forhuman longevity; our lab would like to use deuterium oxide to stabilize the kinesin motorprotein. The kinesin we will be testing are kinesin heavy chain (KHC), also known as kinesin-1and kinesin-5 (Eg5) purchased from Cytoskeleton Inc. Eg5 is different from regular kinesin asinstead of having a cargo binding point, it has two sets of motor domains. The reason for this is,Eg5 is found in mitotic process, needing two motor domains to push to the spindle fibers inopposite directions. Previously in our lab (Koch Lab), graduate students Andy Maloney and

Larry Herskowitz ran a similar stabilizing experiment(Andy Maloney 2010). They useddeionized water, deuterium oxide and oxygen-18 water (water with an oxygen isotope, andhaving the same molecular weight as deuterium oxide as such) attempting to measuring these

isotopic molecules effects on the osmoticpressure changes for displaced waterbetween the microtubule and kinesin motordomain. Using the gliding motility assaythey measured gliding speeds of themicrotubules in each solution. In the glidingassay the kinesin are turned upside-downbound to a passivation layer of casein(Figure 1.3). On top of the kinesin motordomains are placed the microtubules, whichwill glide across the feet of the proteins.However, what they instead measured wasthe viscosity slowing the microtubules

down, as the graphs correlated with theviscosity percentage greater than water. To testour initial hypothesis that deuterium oxide

stabilizes kinesin, we have run, or will run in the future, a series of different stabilization tests.

Figure 1.2A diagram of the gliding motility assay.Image Source: Andy MaloneysDissertation,http://openwetware.org/wiki/User:Andy_Maloney
http://openwetware.org/wiki/User:Andy_Maloneyhttp://openwetware.org/wiki/User:Andy_Maloneyhttp://openwetware.org/wiki/User:Andy_Maloneyhttp://openwetware.org/wiki/User:Andy_Maloney


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The steady state assay, which will involve using the kinesin in the same test every day for a week(or two), and the dynamic light scattering.

The key to the steady state assay is what solution it is being suspended in when it is notbeing used. The kinesin suspended in both deionized water and deuterium oxide, and based onthe hypothesis the kinesin suspended in the deuterium oxide will retain activity longer than the

kinesin in water (see Figure 1.4 for a graphical representation of expected results for a SteadyState test using the gliding motility Assay). We will be using both the ATPase assay as well asthe gliding motility assay mentioned earlier to test the activity of the kinesin.

ATPase Assay

The ATPase assay involves the observations of the ATP hydrolysis of the kinesinmotor domains in real-time. When the ATP drops off the energy to the kinesin motordomains it returns as ADP and an inorganic phosphate. The dye then binds to the releasedADP and a correlation between the amounts of ATP being used over time can be made bymeasuring this increase in absorbance. To do this we use a spectrophotometer, whichmeasures the absorbance of the solution. A decrease in the speed of the absorbanceincrease would indicate a lowering of activity, and no absorbance at all would mean thekinesin has become unstable and denatured. Using the Nanodrop 2000cSpectrophotometer, minute volumes of the sample can measured for maximum efficiency

from a sample (1-2 L). Using small amounts of the kinesin is key to being able to runthe entire Steady State Assay as kinesin is expensive when it isnt grown in the lab.

With the gliding motility assay we will measure the gliding speeds over time. It is understoodbased on the experiment performed by Andy Maloney that the kinesin in deuterium oxide willhave a slower gliding speed than the kinesin in water. However, what we are looking for isgliding consistency, the deuterium suspended kinesin will hopefully stay relatively level inspeed, while the water suspended kinesin will drop dramatically as it begins to destabilize (as

Figure 1.3

A graph representation our expected results of the steady state assay of kinesin suspendedin deuterium oxide and deionized water..

Figure 1.4

A graph of the Bradford Assay practice test. The missing data points are due toreadings the spectrophotometer could not take, but input data is always accepted.


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seen in Figure 1.4). Initially, to run the steady state assay using the ATP hydrolysis analysis theprotein concentrations in solution must be known to get a baseline idea of concentration ofprotein relative to the amount of ATP that is being used. Finding the concentration of protein insolution is found with the Bradford assay. The Bradford Assay works similarly to the ATPaseassay, using an absorbance to concentration correlation. To identify the concentration of theunknown sample a standard with a known concentration must be analyzed first. Separated intofive even standard concentration distributions, a standard curve can be developed. When thestandard curve is made the sample can be analyzed and based on the returned absorbance acorrelation to its concentration can be made. With the Coomassie Dye, the protein concentrationcan be measured through absorbance as the dye binds to the proteins. For practice we usedOvalbumin from chicken egg-whites as both the standard, as well as to test how well thecorrelation worked (results can be seen in Figure 1.5). Though at the time we did not know thekinesin protein would be sent lyophilized, or in a powder. By having the lyophilized kinesin wewould be able to decide what concentration of kinesin we would want in solution. However, theBradford Assay would still be required for the kinesin we had left over in solution from adifferent provider (Dr. Haiqing Liu). Upon arrival we also found out that the expression vector inEg5 were only from 13-437, meaning our protein was truncated missing small parts of both of its

motor domains. Though assured that the kinesin would still work in an assay, until tests are runnothing can be clear about this yet.

DYNAMIC LIGHT SCATTERING

The dynamic light scattering method involves shining a laser at the particles in solution,and measuring the returned diffraction pattern to determine the hydrodynamic radius of theparticle, or the radius of the particle assuming it is a sphere. This is also under the assumptionthat the particles are in Brownian motion (random motion caused by the particles bouncing off ofeach other). Using the returned intensity diffraction the particle size can be found using an auto-

correlation function, which is run through a series of equations. The machine runs all thecalculations and the data outputs the returned intensity and size. The device we used was theDynaPro Titan TC Dynamic Light Scattering Machine (borrowed from Marek Osinskis lab).Over time, as discussed in the steady state assay, the kinesin protein will become unstable overtime. When the protein becomes unstable and denatures, it begins to clump together to formaggregates. Therefore another measure of instability can be identifying increases in aggregatesize, which is why we have been using dynamic light scattering to measure the particles.However, instead of using the output particle size we measure the returned intensity. As theintensity returned is directly related to how large or small the particles are, measuring theintensity does not rely on assumptions about Brownian motion, or spherical shaped particles. Bymeasuring the intensity we also dont have to rely on the machine taking well correlated data

(data that is correlated well is represented by black numbers, while data that is not wellcorrelated comes up in red numbers). Denaturing occurs over time, but can also be inducedthermally. As mentionedin the introduction,kinesin denatures at45C, meaning we canthermally induce theaggregation and measurethe instability quickly

Figure 1.5Difference measure in the time it takes for the particles toaggregate in deuterium oxide and deionized water.

Source: Selfmade.


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over one day instead of over the course of a week. Measuring the returned intensity from theparticles we would expect to see the particles in the deuterium oxide aggregate at a slower at alater time at a higher temperature (See Figure 1.6), much like what occurred with the thermallystabilized fruit flies. We were limited in the amount of kinesin we had, so to practice using thedynamic light scattering machine and looking for optimal settings we used the ovalbumin fromchicken egg-whites protein again, which we bought from Sigma-Aldrich. The ovalbumin workedwell as a practice replacement for the kinesin as ovalbumin also aggregated when heated.Although, the point of thermal instability for ovalbumin was at around 70C to 78C (MireilleWeijers, and Ronald W. Visschers 2002). The experiment I ran was similar parallel to MireilleWeijers and Ronald Visschers experiment of induced aggregation in ovalbumin by heating itfrom 20C to 78C (Our ovalbumin solution did not include NaN3 or NaCl). However, toincorporate our side of the experiment we will be testing the thermal effects on ovalbumin whensuspended in deuterium oxide, deionized water, and deuterium depleted water. Deuteriumdepleted water is being because most natural water contains the slightest bit of deuterium, so tosee if that small amount of deuterium has any effect on the aggregation.

EXPERIMENTAL RESULTS

The initial test run using ovalbumin in only deionized water acted accordingly to our

expectations and the experimental data taking by Weijers. The first concentration we used wasaround 2 mg/mL, however the machine gives errors when a scattering intensity returns at over8,000,000 Cnts/sec and had to reduce the laser power to 5%. The second run of the ovalbuminwas done at a concentration of 200 g/mL at 20% laser power. The lowered laser power was forwhen the protein started aggregating we would still be able to get accurate readings withouthaving to change the laser power mid-experiment, without getting errors. Aggregating at around76C as expecting, using increments of 14C, our first method of data taking involved stoppingdata, changing the temperature and starting the readings again. Though our data came back asexpected there were large gaps of time in between each temperature data set at which we did notreceive any data points and were unsure what was occurring (See Figure 1.7). Due to theamounts of data space that are missed by the incremented time we decided this style of datataking may not be as effective as what we would like considering we dont exactly know howkinesin aggregates, and that is too large of a window to be able to miss critical data.

Testing Deuterium Oxide Stabilization of Ovalbumin (Figure 1.7)

Our new data set consists of data taken from a straight shot from 25C to 90C, todo this we had the set temperature be at 25C and when at 25C, reset it to 90C, but setthe machine to take readings while it was changing its set temperature. We tested past90C because deuterium oxide increases the thermostability. By testing past theaggregation of temperature of ovalbumin, we hoped to see the protein in the deuteriumbegin to aggregate at the much higher temperature at a later time to prove its stabilizingcapabilities. Solutions were made by weighing out 2 mg of lyophilized ovalbumin fromchicken egg-whites and putting it into an aliquot filled with 1 mL of water. This samplewas then filtered with a 0.22 micron filter, and diluted to 200 g/mL in each respectivesolution. The solutions types we tested were ovalbumin suspended in deionized water,90% deuterium oxide, 50% deuterium oxide 50% deionized water, and the original

Figure 1.6A graph representing the aggregation of ovalbumin over time. The final datapoints were calculated based on a ratio of laser power to intensity, as the dynamiclight scattering machine will not read past 8,000,000 Cnts/secs

Figure 1.7Graph of the data for the stability of deionized water against deuterium oxide. 50-50 solution and Extra diluted solution

were included to verify whether there was protein in the deuterium solution and whether aggregation will occur at lessthan 200 g/mL.


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deionized water and protein sample diluted with pure deionized water to make aconcentration of 100 g/mL. The original experimental plan was to only test the 90%deuterium oxide and the deionized water solution. However, as seen on the graph of thedata points (Figure 1.8), the solution with 90% deuterium oxide didnt aggregate at all.Even when we continued to take data points at a steady 90C the intensity remainedrelatively stable other than the small intensity hump, though it cannot be seen in thisdata set. This was unlike what we had expected to happen, as shown in Figure 1.6.To besure the solution with deuterium oxide in it was not missing protein we decided to mix100 L of the water and deuterium solutions, which was approximately 50-50 % in fromthe first set of data (green triangles). Though the intensity water greater for the 50-50solution (which may suggest we were right in our assumption the deuterium solutioncontained no protein, although this is inconclusive) the protein did not aggregate asexpected other than the hump in this data, as well. The new question that came up was,Will ovalbumin even aggregate when its been diluted to that small of a concentration?,assuming the solution of 90% deuterium was in fact empty. To solve this we diluted theoriginal deionized protein solution to a the concentration mentioned above of 100 g/mL.The data shows, the diluted solution did aggregate, but at a delayed time. This would be

expected of a less concentration solution as to properly aggregate the particles must findeach other in solution, if the solution is more dilute the likelihood of the particlesmeeting goes down. Further tests were run to verify if the deuterium oxide solutionactually prevented aggregation all together as even though our run of this would provethat, one can never really be sure.

Although it cannot be seen in the figure above, both solutions containing the deuteriumhad interesting similarities with the intensity trends at the same points in time. By looking at theblown up version of the data trends for the deuterium solution you must note that first, to makethe graphing easier the independent variable for the 50-50% solution is in terms of acquisitions,and because the acquisitions are taken every 5 seconds, simply take the acquisition number andmultiply by 5 to compare the graphs on a timewise function. In both functions the noise

(random data) starts at around the 250 second mark, and when the 500 second mark came iswhen there was the hump in the data. Whether or not this small increase in intensity can beattributed to the aggregation of the ovalbumin cant be said for sure. A speculation can be madethat aggregation occurred but it was suppressed, which would still support the originalhypothesis, as this data hump still occurs at a later time and an increased temperature.

Proceeding with the plan of finding the most effective way to take data, it seemed that astraight shot from 25C to 90C was both too fast to see proper aggregation at a specifictemperature, but also may have an affect on how the protein aggregates when being heated thatquickly. Using a combination of our straight shot method and increment we were able to findan optimal setting for taking data. Setting up an Event Schedule we would set increments of 5degrees starting at 25C, however, we discovered a setting that will take measurements withoutwaiting for the target temperature to be reached first. By taking 20-25 acquisitions per incrementat 5 seconds per acquisition the machine has enough time to move quickly through eachincrement but slows down enough to not be as fast as our data represented in Figure 1.9. Usingthese data settings the next measurements were taken using the solutions, deuterium oxide,deuterium depleted water, and deionized water.

3rd Set of Results (Figure 1.9-2.0)

Figure 1.8

A comparison between the solutions that contained deuterium oxide. Similarities occurring at 250 secs (noise),and 500 secs (beginning of hump).


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The third test was run with the new solutions of 90% deuterium oxide, deionizedwater, and deuterium depleted. The new factor deuterium depleted water being added, asnaturally water has a small amount of deuterium in it and we were testing if thatdeuterium has an effect on the aggregation. We also hoped to replicate the results fromthe 2nd set of results, using completely new samples. The deionized water sample, asexpected aggregated, even with our modified data taking style, although near the end ofour data it began to decrease in intensity, for reasons unknown. As we hoped to see thedeuterium suspended ovalbumin again did not aggregate at all. However, the largestdownside this 3rd set of results was that deuterium depleted solution of protein did notaggregate at all either, which completely went against both our hypothesis and what ourdata had been verifying. Even running the deuterium depleted solution multiple times, itcontinued to come up not aggregating. We thought we have switch up solutions onaccident and put deuterium in the sample that was supposed to be deuterium depleted, sowe used the deuterium depleted water directly from the stock (which should normally notbe done, but we were about run out anyway) and tested the new solution again. Theresults continued to come back negative on the aggregation. Whether or not the machinewas reading the samples right can even be verified by the naked eye as when a solution

aggregates it becomes cloudy, and when it does not it remains clear (Figure 2.1).

Initially our thoughtwas our deuterium depletedsample may have somehowbecome deuterated, or even

contaminated somehow.However, the following

days we proceeded to testthe ovalbumin again and theovalbumin did notaggregate in any solutionwe put it including the puredeionized water solution.What may have happened toour ovalbumin is unclear,however problems with the proteins may be the cause of why the deuterium depleted sample wasnot working properly as it was the last tested sample.

We tested the kinesin next, however we were only able to run our experiment once in one

cuvette run, which meant instead of being able to run the kinesin in both deionized water anddeuterium oxide, we had to pick one or the other. We already knew that kinesin destabilized at45C so we decided to run the kinesin in deuterium up to 60C at laser power 20% again. Thedata we received back was far from conclusive (Figure 2.1).

CONCLUSION

Figure 2.0Non-aggregated sample(left), Aggregated sample(right). The cloudy look onthe left sample is due to condensation.

Figure 1.9Graph of the third data set, testing deuterium oxide, deuterium depleted water anddeionized water. The deuterium depleted water remains un aggregated for unknownreasons.

Figure 2.1A graph of the data received back from running the Eg5 kinesin through the DLS machinefrom 25C to 60C


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Although the ultimate goal of our experiment was not achieved we were able to makesome insights into effectively taking data on the dynamic light scattering machine. Our optimalsettings involved using continuous data taking, while running temperature increments, withacquisitions in between to slow the temperature changing process. Deuterium oxide also seemedto stabilize ovalbumin pretty well as in all the experiments it never really aggregated. The nexttime we run kinesin we also hope to be able to use a smaller concentration of kinesin so we canuse our small sample amount more effectively by making cross comparitive runs instead of justone. We also hope to be able to test more than just the Eg5 kinesin, as we have the kinesin-1(KHC) as well. As mentioned, we have also been testing the kinesin in the steady state assay, buthave not received back full and conclusive data yet.

ACKNOWLEDGEMENTS

Experimental ideas provided by Steve Koch.

Experimental results and testing assistance by Nadiezda Fernandez-Oropeza and Anthony

Salvagno.

Work supported by the funding of the National Science Foundation Research Experience for

Undergraduates, and DTRA under Grant no HDTRA1-09-1-008


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The Ability of Deuterium Oxide to Stabilize the Kinesin Motor Protein