2011 NSF CAREER_Steve Koch Full Project Description

I. Specific Aims We all know that water is essential for life on earth. Despite this, there remain many fundamental questions about how water specifically affects biomolecules and cells. Some of these questions are so important that it is surprising to me that the answers aren’t being pursued with more of our research energy. For example, the brilliant Gilbert Lewis predicted in 1934 that deuterium may be essential for some life formsMendeley Citation{b2df1b3e-26a2-102d-8183-0024e85e2bb9} Prev{1}1, but I cannot find a single peer-reviewed publication from the US investigating this fascinating question. Water isotope effects and osmotic stress studies are also nearly absent from the literature in many important fields, such as with kinesin molecular motors. A final striking example of how many mysteries remain is that almost all of the molecular dynamics simulations for biological molecules either do not explicitly model the water, or are forced to use empirically-derived models for the interactions of water with the biomolecules. Clearly there are many fundamental and key questions about water’s interaction with biomolecules. We will pursue these questions as openly as possible—including publishing this proposal openly. With a primary goal of bringing our discoveries, methods, and ideas to researchers, students, and other curious citizens as quickly as possible, we will maximize the broader impacts of our research to society. Progress in any of these Specific Aims will surely lay a foundation for a productive and exciting research career that I am eager to pursue. 1. Determine whether deuterium is necessary for optimal growth of some living organisms. All naturally-occurring water has about 150 parts per million deuterium, about 17 millimolar concentration. It is a fascinating and open question whether life has evolved a beneficial use for deuterium, perhaps even being essential for some organisms. This hypothesis was originally proposed by Gilbert Lewis in 1934Mendeley Citation{b2df1b3e-26a2-102d-8183-0024e85e2bb9} Prev{1}1 and remains untested! We will test it using plant seeds, E. coli, and yeast. The two sub-aims are: 1.1 Determine whether plant seed germination is slowed or delayed in deuterium-depleted water (DDW). We will continue our preliminary experiments using tobacco seeds and will investigate other types of seeds as well. We will seek to develop germination sensing methods that have the necessary repeatability and precision to detect the expected subtle differences between growth in DDW and ordinary purified water. 1.2 Determine whether growth rates of unicellular organisms are slowed in DDW. We will assess whether cell division rate, colony growth rate, or other measurable parameters are different in DDW relative to ordinary purified water. E. coli and yeast will be the first two organisms we assess. A statistically-significant slower growth in any system would hint towards as of yet unknown mechanisms in cells that have evolved with a preference for deuterium over protium (hydrogen-1). 2. Gain atomistic understanding of water isotope effects and osmotic stress on biomolecule stability and biomolecular interactions. In this aim, we will study water isotope and osmotic stress effects in two systems: kinesin-microtubules and protein-DNA. 2.1 Investigate effects of water isotope and osmotic stress on kinesin stability and activity. Kinesin is a dimeric motor protein that “walks” along microtubules using energy from ATP hydrolysis. Water plays a critical role in the rates of binding and unbinding of the kinesin to / from the microtubule during each step. We will investigate the nature of these interactions with water by assessing kinesin activity, stability, and binding forces as we change the water isotope or the osmotic pressure. 2.2 Investigate role of water in the stability of protein-DNA complexes using single-molecule DNA unzipping. Just as with kinesin, water plays a key role in the binding of proteins to DNA molecules. Site-specific protein-DNA interactions can be probed at the single-molecule level by unzipping DNA molecules with optical tweezers. We will study unbinding forces of Tus, a model protein-DNA system, as we change water isotope or osmotic pressure, revealing information about the hydrating waters excluded from the binding interfaces. 2.3 Use molecular dynamics simulations and the vastly improved charge-transfer (CT) field of our collaborator (Dr. Atlas) to interpret results of aims 2.1 and 2.2 in the context of atomistic water specifically interacting with protein-protein and protein-DNA complexes. In 1995, Parsegian and colleagues stressed that the fundamental importance of water activity is so often overlooked in molecular biologyMendeley Citation{7958f0f4-a550-4ae0-983e-697ded46f8ab} Prev{2}2. In many fields of biophysics today, this is still the case. Aside from our preliminary data with D2O and heavy-oxygen water, we are unaware of any kinesin studies employing water isotopes or osmotic stress. Success in this aim will open up new avenues for understanding how kinesin, DNA-binding proteins, and biomolecules in general are affected by the atomistic interactions of hydrating water molecules. 3. Open science: Open Notebook Science, Open Data. A primary goal of our lab and of me as a scientist is to help transform the practice of science towards a much more open practice than is currently the case. We have been pursuing this goal in both our teaching and our research, for example, practicing Open Notebook Science (ONS) and publishing Open Data. We have discovered that performing science openly feels great and greatly increases the impact of our work. On the other hand, we have also discovered that sharing efficiently, rapidly, and reliably is not easy and requires collaboration with experts such as library information scientists. Many areas of Open Science are very new and thus optimal methods need to be discovered through research projects of their own. In this Specific Aim, we will work with our collaborators on concrete Open Data and Open Notebook Science projects to reliably share the science of our laboratory and in doing so develop open science methods that can be replicated and improved upon by other researchers around the globe. II. Background and significance A. Open Science. Lack of open sharing of data, methods, and ideas is currently slowing research progress and minimizing impacts of global research efforts. There are two main causes of this problem. First, the reward system in the federally-funded research system forces most scientists to only partially share information and to hoard data, expertise, software, ideas, etc. in order to protect funding prospects, job security, etc. In most fields, it’s currently a “share at your own risk” system. The second problem is that research labs generate a huge amount and variety of things that would be useful to share, but there is limited time, limited infrastructure, and limited expertise to do so. In many fields sharing efficiently, reliably, and effectively is not easy. It is my belief that the broader impacts of my teaching and research is best maximized by sharing ideas, methods, failed results, successes, and publications as openly, quickly, and efficiently as possible. A growing number of scientists worldwide share this opinion and an quot;
open sciencequot;
movement has been gaining momentum quickly during my career as a PI. In my research and education, we’ve made a lot of progress in Open Notebook Science, Open Data, Open Publishing, Open Source—and we’ve been delighted with the results. We’re convinced that we’re on the right path with open science, and funding agencies such as the NSF agree and are steadily improving the incentives for openness. So, the first problem described above is solved as far as my research and education career are concerned. But the second problem—carrying out Open Science effectively and efficiently remains a hurdle for us and for others. In our proposed research, we will take on this second problem and developing re-usable methods for open science in our field of molecular biophysics. Current state of open science. Progress is being made too quickly to describe the current state of the open science movement. There are also too many bold leaders and innovators to name individually, though many of these people are very active in online communites (e.g. http://friendfeed.com/science-2-0) An excellent example of the power of open science is provided by Jean-Claude Bradley, Andy Lang, and their collaborators (http://onschallenge.wikispaces.com/). The nature of the science in our biophysics lab necessitates different methods that we will develop during this proposed research. Open Notebook Science in Junior Lab: Students in the undergraduate modern physics lab course I teach carry out their work completely openly using Open Notebook Science. We described this successful endeavor in a recent book chapterMendeley Citation{a9895ded-3f24-4168-a3c8-44181c903004} Prev{3}3. B. Deuterium in living cells. About 1 out of every 6,600 hydrogen atoms in naturally occurring water are the isotope deuterium (D; Hydrogen-2; one proton, one neutron). Because the nucleus of deuterium is about twice as massive as protium (Hydrogen-1), the chemistry of D is significantly different from H. D-bonds have a lower ground state energy than H-bonds and thus have a longer lifetime (e.g. D-C versus H-C). In water, deuterium changes the hydrogen bonding environment and significantly affects the behavior of solutes (known as a solvent isotope effect). Heavy water is water that has almost all D2O —it is 25% more viscous than regular water, freezes just under 4°C, is about 10% denser than H2O, and is toxic to living cellsMendeley Citation{5acd745c-a5ae-4b68-b040-764e8efa099b} Prev{4}4. This last fact—that deuterium is toxic to cells—greatly surprised me when I learned it a few years ago. But in the early 1930’s when deuterium was first detected, many scientists, including Gilbert Lewis realized this would be the caseMendeley Citation{5acd745c-a5ae-4b68-b040-764e8efa099b};{a8a33010-531b-4c02-b40d-81a186f9e048} Prev{4,5}4,5. In 1933, Lewis first demonstrated the toxicity by showing that tobacco seeds did not germinate in D2O, results he reported in a simple and fascinating paper in JACSMendeley Citation{5acd745c-a5ae-4b68-b040-764e8efa099b} Prev{4}4. Despite the labor involved in purifying D2O, many other results by Lewis and others quickly followed, showing toxicity of deuterium-enriched water to all living systems studied. In the following decades, D2O became widely available and inexpensive and much has been learned about the behavior of cells and biomolecules in deuterium-enriched waterMendeley Citation{51ad7e64-11c4-4270-be3a-658b568ab3b4} Prev{6}6. Because of its effect on hydrogen bonding, D2O tends to stabilize proteins and macromolecular structures. D2O significantly stabilizes microtubulesMendeley Citation{7418604e-c721-42dc-8abd-57a6ae805601} Prev{7}7, much like the antimitotic drug taxol, and this is the primary reason that D2O is toxic to eukaryotic cellsMendeley Citation{aac31099-c886-4d3a-a777-f7bb8ba59281} Prev{8}8. This stabilization seems to a general effect of D2O and often manifests as an increased thermostability of proteinsMendeley Citation{51ad7e64-11c4-4270-be3a-658b568ab3b4} Prev{6}6. Tubulin (the building block of microtubules) is notoriously unstable in H2O, even at 4°C, but is much more stable in D2O Mendeley Citation{1610ffba-e5a6-4ef0-9e57-d9f13f0af3c2} Prev{9}9. D2O is actively pursued as a means for stabilizing vaccines for transport into locations without refrigeration. D2O has even been shown to confer whole-organism thermostability on deuterated fruit fliesMendeley Citation{aed8d3aa-20a7-102d-8183-0024e85e2bb9} Prev{10}10. While there are not as many studies on the biological effects of deuterium-enriched water as I had expected, it’s fair to say that much is known about the subject. In stark contrast, there are almost no reliable results published on the effects of deuterium-depleted water in biology. As of July 2011, a Pub Med search for “deuterium depleted” OR “deuterium depletion” yields only 7 results pertaining to effects on living cells. I can’t explain why this is the case, especially considering how Lewis ended in his 1934 letter to Science magazine titled, “The Biology of Heavy Water”: “It is not inconceivable that heavy hydrogen, which exists in small amounts in all natural water, may actually be essential to some plants or animals. A supply of water almost completely freed from the heavy isotope is now being prepared for the purpose of conducting such studies.”Mendeley Citation{b2df1b3e-26a2-102d-8183-0024e85e2bb9} Prev{1}1 I could not find published results or further mention of the deuterium-depletion studies Lewis mentioned. Perhaps the experiments provided “negative” results and thus were not deemed worthy of publication? Perhaps the deuterium-enrichment studies were too engaging? Whatever the case, I can’t explain the dearth of published findings in the subsequent 75 years. I have found only a few published reports. These indicate that deuterium-depleted water (DDW) slowed the growth of cancer cells and reversed tumor growth in miceMendeley Citation{dcd8b65a-6a70-42a8-b4ba-b87b3885688c} Prev{11}11. Alarmingly, while Pub Med provides only a handful of deuterium-depletion resultsMendeley Citation{dcd8b65a-6a70-42a8-b4ba-b87b3885688c};{904ef269-a5e9-420e-9685-b9ec8de7d0c1};{c78c359b-1aa5-4953-9661-ce45f9718672} Prev{11-13}11-13, Google results are dominated by thousands of webpages describing DDW as a cure for cancer and many other ailments. Obviously there is a huge disconnect between the research and the proposed therapies. The research is so lacking that even though I have published no deuterium research papers, I have already been contacted by a patient seeking advice about DDW as a potential therapyMendeley Citation{8aeed90a-094d-46ba-9b83-def69c5fa96a} Prev{14}14. The question Lewis proposed seems clearly unanswered and is fundamental and fascinating. 150 parts per million deuterium in natural water may seem small, but, from a biochemical viewpoint, it amounts to about a 17 millimolar “impurity” in what we usually consider “pure” H2O. Cells have complicated molecular motors that pump protons (H-1) across membranes, or use proton gradients to generate mechanical motion. It’s conceivable that pumps or motors specific for deuterium have evolved in some organisms. Certainly when the molecular machines we know about encounter D instead of H they should behave differently. Can these rare effects be seen in single-molecule studies, for example via molecular motor stalling? If they aren’t seen, why not? Do cells grow differently in deuterium-depleted water? Whatever the answer: yes (slower / faster) or no—the answer is important and the question begs to be investigated. C. Effects of water on biomolecular interactions. In many fields of biophysics, the importance of water is often overlooked. In 1995, Parsegian, Rand, and Rau highlighted this problem in an excellent methods paper, “Macromolecules and Water: Probing with Osmotic Stress,” where they say: “Even though there is so much of it, and even though its activity is so carefully controlled in nature, water is usually taken for granted…For every thousand papers on enzyme reactions or ligand binding that carefully list ligand activity, pH, temperature, salt concentrations, and solute composition, there is probably not one that notes explicitly the activity of this numerically and functionally most essential species.”Mendeley Citation{7958f0f4-a550-4ae0-983e-697ded46f8ab} Prev{2}2 38366702959100Protein and DNA molecules are profoundly impacted by their solvent, water. Water molecules form “hydration layers” around these solutes, characterized by a more ordered water structure with slower dynamics compared to the “bulk water” molecules that are far away from solutes or surfacesMendeley Citation{9b4b5778-47da-4326-bc94-3f9987597797};{d5802f97-940f-41be-8f42-b8b7af6abb7a} Prev{15,16}15,16. When two biomolecules form a bound complex (for example protein-DNA binding), their overall surface area usually decreases by an amount related to the area of their binding surfaces. Dozens to hundreds of “hydrating” water molecules are excluded from this binding interface and return to the “bulk” water. If the chemical potential of the bulk water is reduced, for example by adding macromolecular osmotic stress agents (increasing the osmotic pressure), then it is more energetically favorable for molecules to bind and produce more “bulk” water molecules. By studying how the binding equilibrium varies as a function of water osmotic pressure, the number of water molecules excluded from the binding interface can be deducedMendeley Citation{7958f0f4-a550-4ae0-983e-697ded46f8ab} Prev{2}2. Similarly, the number of water molecules exchanged upon binding / unbinding can be measured by measuring the unbinding rate of the complexMendeley Citation{52f69c88-e8d1-102c-9b04-0024e85ead87} Prev{17}17. These thermodynamic relations are important for a number of reasons. First, they show how dramatically water affects biomolecular interactions. For a typical bi-molecular interaction, such as protein-DNA or kinesin-microtubule, a hundred or more water molecules can be excluded from the binding surfaces. As shown by Sidorova and Rau, the chemical potential of the bulk water can be changed by adding small molecules such as betaine or sucrose to concentrations greater than 3 molar, resulting in a binding lifetime increased by more than 3 orders of magnitudeMendeley Citation{52f69c88-e8d1-102c-9b04-0024e85ead87};{1c82bab1-76ee-48ee-bb96-5207d660e358} Prev{17,18}17,18. Secondly, these thermodynamic relations provide a means for specifically studying the effect of water molecules at the biomolecular binding interfaces. Single-molecule protein-DNA by unzipping 33058102192655In my dissertation work, I showed that positions of DNA-binding proteins could be mapped with high resolution by unzipping single DNA molecules with optical tweezersMendeley Citation{47ab3a5a-e8d1-102c-9b04-0024e85ead87} Prev{19}19. Fig. 1 shows conceptually how these experiments are carried out with optical tweezers. To enable these experiments, I designed and implemented a versatile unzipping anchoring construct that allows for unzipping of any DNA molecule with a known 5’ or 3’ overhang (see Fig. 2). The method and DNA construct have been successfully used for investigating DNA repair proteinsMendeley Citation{aedf17af-1dde-41d3-a5ec-d78fc6e4ea47} Prev{20}20, helicaseMendeley Citation{2e1e930c-e8d1-102c-9b04-0024e85ead87} Prev{21}21, nucleosomesMendeley Citation{3f68482b-54c2-422d-ac70-ea04828ac93f};{1b84dac6-4ac5-4c68-a5f4-b834f83054cc} Prev{22,23}22,23, and other proteins. Beyond mapping, I also showed that the site-specific protein unbinding rates could be probed via the dynamic force spectroscopy method of Evans and RitchieMendeley Citation{62847e92-bc17-4e86-be13-5c7ff8add379};{7c0bef63-cbaf-452c-86c7-4a0681124e09};{7321813d-425c-42eb-91ce-302d5a27b500} Prev{24-26}24-26. In this proposal, we will leverage these capabilities to probe the effect of water isotope and osmotic stress on Tus protein-DNA interactions. Tus protein There are many well-studied protein-DNA systems that we can explore via DNA unzipping. The one we wish to pursue is Tus protein, due to its importance in prokaryotic DNA replication and because we can obtain purified protein and many mutants via our collaborator Dr. Cameron Neylon. Tus binds to asymmetric sites on DNA and only blocks replication fork progression in one orientationMendeley Citation{788c18b0-fa34-448e-b121-de0e7786d741};{0ab9f173-d7e0-4d3a-addb-11413694b25b} Prev{27,28}27,28. Thus, in addition to providing an interesting model system for our water studies, it is particularly interesting in single-molecule unzipping studies, which may mimic the behavior of unzipping fork progression. D. Robust Kinesin Gliding Motility Assay. We have spent the past two years perfecting the kinesin gliding motility assay and now have an almost fully-automated system for rapidly assessing kinesin activity under various buffer conditions. The work has been described extensively in Andy Maloney’s Open Science dissertationMendeley Citation{29b859ac-454a-4b2d-ae29-4137143ca2da} Prev{29}29 ( HYPERLINK quot;
http://www.openwetware.org/wiki/User:Andy_Maloneyquot;
quot;
Dissertationquot;
http://www.openwetware.org/wiki/User:Andy_Maloney#Dissertation and related publicationsMendeley Citation{3388b370-0f8f-465c-afb8-29a415a34051} Prev{30}30. From “freezer to figure,” our gliding assay system allows us to measure the speed of hundreds of microtubules under one assay condition in under 4 hours. III. Preliminary Studies A. Open Science. Open Data in the Research Laboratory: To date, our most formal and well thought out open science study has been our collaboration with Dr. Rob Olendorf, a professor in the UNM library. We have undergone a pilot project to archive all of the data associated with our recent PLoS ONE publication on the affects of casein in gliding motility assaysMendeley Citation{3388b370-0f8f-465c-afb8-29a415a34051} Prev{30}30—Including electronic notebook entries, our open-source image tracking and speed analysis software, raw image data, and all levels of processed data. At the time of this writing, most of the work is computational and being carried out by Dr. Olendorf, but a finished, citable data archive is not yet available. What we do have are the data sets for the publication hosted on our own server at www.kochlab.org/files/passivation. These data are potentially valuables for readers of our open access publication, but their utility and discoverability is severely limited by lack of metadata. Furthermore, we do not consider our lab server a suitable perpetual archive of our data. These problems will be eliminated by successful completion of our data curation / archiving project with Dr. Olendorf and the UNM Library. We also publish much of our image series data on youtube manually, along with an “open data” license and description of the gliding assays. Despite being far from a perfect form of data sharing, we did enjoy a wonderful success from doing so. Only a couple months after we posted it, researchers at UC Santa Cruz discovered our open data via Google searching and re-used our data in a publication of their own for a purpose that we had never consideredMendeley Citation{93a6f007-7402-456d-b14c-8de714e9d193} Prev{31}31. While a small-scale example, this was a wonderful example of the power of open, freely reusable, and discoverable data. 362013556515B. Deuterium-depletion As a side project during his dissertation work, our former Ph.D. student Andy Maloney began replicating Lewis’s 1930’s experiments with tobacco seeds and heavy water. At the advice of a colleague on FriendFeed, we purchased tobacco seeds from a supplier in the UK, “The Tobacco Seed Company.” As shown in Fig. 3, we were easily able to replicate Lewis’s 1933 results and showed that the seeds reliably germinated in distilled and tap water, but none germinated in 99% pure D2O. We also attempted growth in deuterium-depleted water and we detected growth but did not determine whether it was slower, faster, or the same as in regular water. We spent considerable time designing a webcam system so that the experiments could be viewed in real-time on the internet. This is a nice system that we may utilize, but I am not sure that it is the best way forward. From these preliminary experiments, we learned that the seeds do not sit still when sprouting and measuring sprout length is probably not precise enough to detect differences from deuterium-depleted water. We will redesign the experiments so we can more precisely measure “time to germination” or other factors that may have less variability from seed to seed. An example of a type of measurement we may perform is shown in Fig. 4. When tobacco seeds are viewed with a 10x microscope objective, the region of sprouting can easily be seen (see Fig. 5). Assessing sprouting via microscopy or macro photography may offer good route for discerning more subtle differences in growth. We do not have preliminary work with yeast or E. coli, but a number of our colleagues at UNM routinely grow these organisms and we have our own prior experience using E. coli to produce kinesin protein and plasmid DNA. C. Single-molecule work: Optical tweezers and DNA unzipping Optical tweezers instrumentation; Data acquisition and analysis software We have constructed a single-beam optical trapping system that can exert forces greater than 100 piconewtons on 0.5 micron polystyrene microspheres. Key hardware elements of our system are a 2 watt diode-pumped solid state laser from Crystalaser; acousto-optic modulator (AOM) from Gooch and Housego (formerly NEOS); a custom-order high-IR transmission 60x water immersion PLANAPO objective from Olympus; and Olympus IX-71 microscope body; 1-D piezo stage from Mad City Labs for horizontal displacement of sample; 1-D piezo objective piezo mount from Mad City Labs for vertical trap displacement; quadrant photodiode and amplifier from On-Trak; 4 independent low pass analog filters from Krohn-Hite; multiplexed data acquisition cards from National Instruments. There are two critical software components of our optical tweezers system. The first is the optical tweezers feedback control softwarePEVuZE5vdGU+PENpdGU+PFJlY051bT43MTM8L1JlY051bT48cmVjb3JkPjxyZWMtbnVtYmVyPjcx
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ADDIN EN.CITE.DATA (5, 56), which we have succeeded in upgrading to the newer DAQmx version of LabVIEW. This software provides the necessary feedback modules (force clamp; velocity clamp; loading rate clamp) for DNA unzipping and protein-DNA unzipping. The other critical software component is a suite of automated data analysis LabVIEW programs. These programs convert the raw OT data into “force v. unzipping index,” and are further automated to identify locations and disruption forces of DNA binding proteins ADDIN EN.CITE <EndNote><Cite><Author>Koch</Author><Year>2003</Year><RecNum>17</RecNum><record><rec-number>17</rec-number><foreign-keys><key app=quot;
ENquot;
db-id=quot;
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>17</key></foreign-keys><ref-type name=quot;
Journal Articlequot;
>17</ref-type><contributors><authors><author>Koch, S. J.</author><author>Wang, M. D.</author></authors></contributors><auth-address>Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853, USA.</auth-address><titles><title>Dynamic force spectroscopy of protein-DNA interactions by unzipping DNA</title><secondary-title>Phys Rev Lett</secondary-title></titles><periodical><full-title>Phys Rev Lett</full-title></periodical><pages>028103</pages><volume>91</volume><number>2</number><keywords><keyword>DNA/*chemistry/metabolism</keyword><keyword>Deoxyribonucleases, Type II Site-Specific/*chemistry/metabolism</keyword><keyword>Kinetics</keyword><keyword>Spectrum Analysis/methods</keyword><keyword>Substrate Specificity</keyword><keyword>Support, Non-U.S. Gov't</keyword><keyword>Support, U.S. Gov't, P.H.S.</keyword><keyword>Thermodynamics</keyword></keywords><dates><year>2003</year><pub-dates><date>Jul 11</date></pub-dates></dates><accession-num>12906513</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12906513</url><url>file://C:%5CDocuments%20and%20Settings%5Csjkoch%5CMy%20Documents%5CReferences%5CFriends,%20Me%5CKoch%20and%20Wang_UFAPA%20Kinetics.pdf</url></related-urls></urls></record></Cite></EndNote>(6). The suite has been upgraded to LabVIEW 8.5. The primary coders of the original software applications were the PI (Koch) and his colleague Dr. Richard Yeh during their Ph.D work. Construction of our optical tweezers system has taken much longer than expected, due to lack of funding, some bad luck with expensive lasers, and my own mis-steps. However, at this point we have working system with excellent control software. We have proven that we have sufficient power and trap quality to easily overstretch single DNA molecules, which is known to require about 65 piconewtons of forceMendeley Citation{7e219628-d2c3-4634-9474-196eb6f6a67c};{de6337be-69d7-4543-80bb-39802c5c1d39} Prev{32,33}32,33. DNA Unzipping As described in Background and Significance, I have extensive experience in DNA unzipping from my work in graduate school. At the time, producing a DNA construct capable of unzipping took me quite a while, but once I did, I was able to produce a number of different constructs easily. Subsequent graduate students were also able to use these methods. Despite my expertise, graduate students in our current lab have yet to produce constructs that unzip more than 3% of the time. The constructs look fine via gel analysisMendeley Citation{0d17a975-db55-4bcb-8b10-4c3ade74f6f3} Prev{34}34, but for still unknown reasons the tethers in our samples are 10 times more likely to overstretch than to unzip, preventing us from carrying out experiments reliably. Our previous NSF CAREER proposal was focused on DNA unzipping, and because of our failure to produce the constructs, we have greatly reduced our focus on DNA unzipping for this proposal. Nonetheless, I believe if funded we can surmount this hurdle. DNA Unzipping will allow us to probe the affects of water on site-specific protein-DNA complexes at the single-molecule level. My previous work showed that the unbinding rate can be probed via unzipping. Preliminary work shows that osmotic stress systematically increases the unbinding forces (Fig. 6). Thus, if we can reliably create unzipping constructs, we will be able to develop a new method for probing site-specific protein-DNA interactions at the single-molecule level as we change water isotope and osmotic pressure. D. Isotope and Osmotic Stress effects in kinesin gliding assays. Water Isotope: As shown in Fig. 7, heavy-hydrogen water (D2O) and heavy-oxygen water (H2O-18) systematically slow microtubule gliding speedsMendeley Citation{29b859ac-454a-4b2d-ae29-4137143ca2da} Prev{29}29. We first observed the change from D2O and suspected that the effect was possibly due to the common general slowing of chemical reactions involving D-bonds, a kinetic isotope effect. However, when we performed the much more expensive oxygen-18 experiments, we were surprised to also find a small but significant slow-down. Furthermore, both data sets are fit well by a simple linear fit—indicating that at least one of the rate-limiting steps is being affected. Interpretation of these data are part of specific aim 2.1 of this proposal. We have noted that the relative affects of heavy-hydrogen and heavy-oxygen are similar to the relative changes in viscosity of the two solvents. We will investigate how this could translate to the observed speed changes. Osmotic stress: We have seen (data not shown) that moderate osmotic stress—up to 2 M betaine—strongly reduces microtubule gliding speed by more than 75%. Thus, we can probe the effect of water on kinesin-microtubule interactions via small molecule osmolytes. As with solvent isotope, we will need to couple experiments, kinetic modeling, and atomistic simulations to understand how this speed change is brought about by specific water interactions at the binding interfaces. IV. Research Methods Specific Aim 1: Determine whether deuterium is necessary for optimal growth of some living organisms Sub Aim 1.1: Plant seed germination in DDW. Following the lead of Lewis, our preliminary work has been performed with the very accessible system of tobacco seeds. Lewis’ colleague presumably recommended this system for good reasons: These seeds germinate reliably in ordinary distilled water at a rate approaching 100%. The seeds are also small, so when placed in small volumes of water, they will not perturb the water too much, even if it is assumed that all of the naturally-existing hydrogens in the seed exchange with the surrounding buffer. Finally, due to the industrial importance, high quality seeds are inexpensive and readily available. Admittedly, we are not experts in any kind of plant biology. Thus, a significant part of the research project will involve identifying and communicating with experts who can guide us towards optimal methods. Furthermore, the graduate student leading this project will become an expert in modern methods for assessing seed growth. As of now, our research plan is the following: ,[object Object]

[optional] pre-soak seeds in the experimental buffer for several days in the dark at 5°C. The dark and cold will prevent germination, while allowing for any equilibration of hydrogens in the seeds with the surrounding buffer. The purpose of this step is to avoid any germination that may occur at room temperature before the buffer water has equilibrated with the seeds, and may reduce seed-to-seed variability and improve our signal to noise ratio.

Exhange seeds to fresh buffer at room temperature and uniform lighting. We will continue to use standard spectroscopy cuvettes sealed with parafilm or possibly covered with mineral oil to prevent exchange with atmospheric water.

Periodically assess germination or sprouting via various methods: continuous webcam photography; manual DSLR photography with macro lens; or manual microscopy with 10x magnification.We will surely need to adapt and optimize the above protocol in order to detect expected differences in deuterium-depleted water. Furthermore, tobacco seeds are unlikely to be the optimal system to study. We will consult with experts and choose several other plant varieties to examine. Small seeds that reliably germinate in distilled water are ideal. One obvious organism that comes to mind is Arabidopsis, since it is a model organism and has a sequenced and well-annotated genome. Broader Impacts Note: These experiments, especially the seed-germination experiments are especially accessible to younger scientists performing research for middle-school science fairs. Even the most specialized components, D2O and DDW, are not prohibitively expensive and there are an almost endless-variety of experiments that students could pursue. These could potentially displace the “do beans sprout in kool aid” experiments that currently remain popular. Sub Aim 1.2: Determine whether growth rates of unicellular organisms are slowed in DDW. E. coli and baker’s yeast are model organisms widely used on our campus. In consultation with experts, we will design experiments with optimal ability to detect differences in growth rate in these organisms. We have three methods in mind now: (1) inoculate liquid cultures and assess growth or doubling time via spectrophotometry, (2) observe colony growth on agar plates, or (3) observe individual cells directly via optical microscopy. Due to difficulty in preparing growth media that is reliably deuterium-depleted, I believe that method (3) is the most likely method we will pursue. We have had previous collaborations with Dr. Mary Ann Osley and Dr. Kelly Trujillo, experts in yeast genetics, though we have yet to discuss these experiments with them. Flow cytometry, a strength of UNM, is another avenue that would potentially provide a means for assessing differences in cell cycles or cell growth due to deuterium depletion. Specific Aim 2: Gain atomistic understanding of water isotope effects and osmotic stress on biomolecule stability and biomolecular interactions Sub Aim 2.1: Investigate effects of water isotope and osmotic stress on kinesin stability and activity. Building on the results described in the preliminary work, we will measure the speed of kinesin under a variety of different buffer conditions: (a) water isotope—these data have already been acquired for wild-type kinesin-1. We will pursue similar experiments with other kinesin varieties (such as Eg5) and with mutant kinesins we design that may have different interactions with water molecules as predicted by our collaboration with the Atlas lab and atomistic kinesin simulations; (b) osmotic stress—we will measure gliding speed using a variety of osmotic stress agents such as betaine, sucrose, and proline. At a given osmotic pressure, these osmolytes have very different solution viscosities. Thus, we can discern the differing effect of solution viscosity versus osmotic pressure; (c) Using dynamic light scattering and optical microscopy, we will investigate the stabilizing effects of heavy water and osmotic stress agents on kinesin stability at 4C and room temperature. Sub Aim 2.2: Investigate role of water in the stability of protein-DNA complexes using single-molecule DNA unzipping. We will measure the unzipping forces of Tus-Ter complexes under varying loading rates and in a number of different solvent conditions—heavy water or increased osmotic pressure. Each condition will be analyzed with Dynamic Force Spectroscopy (DFS) to estimate the site-specific lifetime of the bound complex. Variation of this lifetime as the solvent is modified will indicate the number of hydrating water molecules. The experiments will be repeated with mutant Tus protein and the difference in hydrating water molecules will be determined. Sub Aim 2.3: Use molecular dynamics simulations and the vastly improved charge-transfer (CT) field of our collaborator (Dr. Atlas) to interpret results of aims 2.1 and 2.2 in the context of atomistic water specifically interacting with protein-protein and protein-DNA complexes. I am currently co-PI on a DTRA-funded basic research project with Dr. Susan Atlas as the PI. As part of this research, the Atlas lab has developed a CT force field and a molecular dynamics parallel code (pdQ) that for the first time models the interaction of water molecules from first principles (as opposed to empirical fitting). During this research, the Atlas lab will use the CT force field to simulate the dynamics of the molecular motor kinesin, it’s tubulin binding site, and the explicit water molecules at the binding interfaces. This research will directly couple to specific aim 2.1 above. As part of this NSF CAREER proposal, we will fund ½ of a graduate student’s time to spend adapting the kinesin MD code for the purpose of modeling Tus-Ter protein-DNA complexes in support of specific aim 2.2. This student will interact with students in Dr. Atlas’ lab to learn the MD techniques, but unless further outside funding for our collaboration is obtained, we will not have a full-time shared MD student for the protein-DNA modeling. Specific Aim 3: Open science: Open Notebook Science, Open Data. As described throughout this proposal, we are commited to making our research and education as openly available as possible. Some steps to do this are easy, such as utilizing Open Access publishing such as PLoS ONE. However, much work remains to efficiently and effectively carry out Open Notebook Science and Open Data. By the time this research plan is funded, the available tools and knowhow will most likely have changed dramatically. Thus, we do not have a detailed plan for our research into open science methods. The most important part of our plan is that we will work with experts, especially collaborators who are experts in library and information sciences. Our main partner is Dr. Rob Olendorf a newly-hired data curation scientist with the UNM library. We are currently working with Rob on curation and archival of our gliding motility assay data, software, and notebooks. We will leverage these experiences in this Specific Aim to improve our data archiving and sharing methods and will openly publish our challenges and successes along the way. V. Educational Integration and Broader Impacts Our lab believes strongly in our mission of advancing molecular cell biology, and therefore our goals are well aligned with those of the NSF for integrating research with education and maximizing the broader impacts of our research. Broadly communicating our results and methods, teaching at all levels, and training future leaders in biophysics research together will greatly multiply the impact of our research discoveries. Below, we describe our plans towards this goal in two areas: open science and integration of research with undergraduate education. One way of broadening our impact is in recruitment and training of underrepresented minorities to biophysics, and this is an underlying component of our goals below. We are at an advantage in this area, because of the unique demographics of the University of New Mexico and the surrounding area. UNM is designated a Hispanic-Serving Institution by the US Dept. Ed. and other government agencies. We participate in the UNM PREP program that recruits minorities to science and our lab has employed three Hispanic researchers so far. Additionally, the local population consists of a high proportion of minorities underrepresented in science. I have participated in community scientific outreach since early in my graduate career and it remains an enjoyable and valued activity. During my first three years at UNM, I have judged the Central New Mexico Science and Engineering Challenge (Middle School Microbiology), the Cleveland Middle School Science Fair, and given a presentation on nanomanipulation in the biological sciences to local Middle and High School science teachers at the NNIN-sponsored summer “nanocamp.” I plan to grow my own outreach, and continue to encourage, value, and reward community outreach by the graduate, undergraduate, and postdoctoral members of our lab. Open Science Since early 2007, our lab has been actively participating in OpenWetWare (OWW), an Open Science site hosted by MIT (http://www.openwetware.org/wiki/Koch_Lab). The goal of open science is complete open sharing of all data and knowledge generated in the laboratory. As our lab has matured, we have taken many steps and a few leaps towards open science. These include “open notebook science” via OpenWetWare (http://openwetware.org/wiki/Koch_Lab:Notebooks), detailed protocol publishing, and some fully-open research proposals. If funded, we are committed to carrying out the proposed research as openly as possible. This will include Open Notebook Science, Open Access publishing, free availability of raw data and all stages of processed data, and sharing of all data acquisition and processing software as described in Specific Aim 3. Our ability to succeed in our open science goals will be greatly aided by the CAREER award and the stability and assurance it will provide our lab. Integration of research with university education. One course I teach is Junior Lab, which is the first “real” physics lab course that physics majors at UNM enroll in. The main innovation with this course is that I host this course completely as Open Science, on OpenWetWare (http://openwetware.org/wiki/Physics307L). The students keep all of their notes electronically on OWW, along with their lab write-ups and formal reports. Additionally, all of the written feedback from me (with the exception of letter grades) is also carried out in public on OWW. The results have been very rewarding, overwhelming positive feedback from students and several students have continued to use ONS in their futher research studies. I think there are significant benefits to training students in open science at this early stage in their research careers and will ultimately make a large impact on the next generation of open scientists. In addition to the open science component to Junior Lab, I intend to add new biophysics modules to the course. Specific modules I would like to add are: tethered particle motion (TPM) analysis of single DNA molecules optical tweezers / magnetic tweezers experiments, and kinetic modeling of enzymatic reactions. I have commitment from the chair of UNM Physics Dept. to support our goals of adding biophysics experiment modules with college and department funds when needed (see Chair letter). Another course I teach is in Biomedical Engineering (BME) and is a course originally developed by Dr. Evan Evans, titled, “Mechanics and Thermodynamics of Molecular Components of Cells. A majority of the topics in the course are directly relevant to this proposal, such as osmotic stress effects on biomolecular interactionsMendeley Citation{7958f0f4-a550-4ae0-983e-697ded46f8ab} Prev{2}2, Kramers’ reaction rate theoryMendeley Citation{5b208a3d-8224-4c9e-a0a5-53dd7ddefb22} Prev{35}35, and single-molecule bond lifetime under forceMendeley Citation{7c0bef63-cbaf-452c-86c7-4a0681124e09};{7321813d-425c-42eb-91ce-302d5a27b500} Prev{25,26}25,26. More importantly, however, I also stress open science methods in this graduate-level course. Most of the students in this course are Ph.D. trainees and use of open science can directly impact their own dissertation research if they choose. Two examples of open science in this course are (1) DNA unzipping data analysis homework assignment, where the students are required to upload their analyzed data sets and figures to FigShare (www.figshare.com), and open data sharing site, and (2) a lab experiment where students perform a gliding motility assay and must post their data and analyses on FigShare. Examples from Spring 2011 students in this latter exercise can be found on the following link: http://figshare.com/figures/index.php/MTC2011Gliding. I loved seeing the students perform these open science assignments and learn some specific Open Data methods. Moreover, the experiments they performed were novel experiments (gliding assays performed with microtubules polymerized in D2O), so there is a chance their open data will benefit current kinesin researchers. References Cited Mendeley Bibliography1.Lewis, G.N. THE BIOLOGY OF HEAVY WATER. Science (New York, N.Y.) 79, 151-153(1934). 2.Parsegian, V.A., Rand, R.P. & Rau, D.C. Macromolecules and water: probing with osmotic stress. Methods in enzymology 259, (1995). 3.Bradley, J.-C. et al. Collaboration Using Open Notebook Science in Academia. Collaborative Computational Technologies for Biomedical Research (2011). 4.Lewis, G.N. The biochemistry of water containing hydrogen isotope. Journal of the American Chemical Society 55, 3503–3504(1933). 5.Taylor, H.S. et al. The Effect of Water Containing the Isotope of Hydrogen upon Fresh Water Organisms. The Journal of Chemical Physics 1, 751(1933). 6.Sen, A. et al. Role of heavy water in biological sciences with an emphasis on thermostabilization of vaccines. Expert review of vaccines 8, 1587-602(2009). 7.Itoh, T. & Sato, H. The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro. Biochimica et biophysica acta 800, 21(1984). 8.Gross, P.R. & Spindel, W. HEAVY WATER INHIBITION OF CELL DIVISION: AN APPROACH TO MECHANISM•. Annals of the New York Academy of Sciences 90, 500–522(1960). 9.Chakrabarti, G. et al. Stabilization of tubulin by deuterium oxide. Biochemistry 38, 3067-72(1999). 10.Pittendrigh, C.S. & Cosbey, E.S. On the Very Rapid Enhancement by D2O of the Temperature-Tolerance of Adult Drosophila. Proceedings of the National Academy of Sciences of the United States of America 71, 540-543(1974). 11.Somlyai, G. et al. Naturally occurring deuterium is essential for the normal growth rate of cells. FEBS letters 317, 1-4(1993). 12.Krempels, K., Somlyai, I. & Somlyai, G. A retrospective evaluation of the effects of deuterium depleted water consumption on 4 patients with brain metastases from lung cancer. Integrative cancer therapies 7, 172-81(2008). 13.KATZ, J.J. et al. Some observations on biological effects of deuterium, with special reference to effects on neoplastic processes. Journal of the National Cancer Institute 18, 641-59(1957). 14.Koch, S.J. I am maximally-skeptical that there currently exists any evidence that drinking deuterium-depleted water has health benefits or will cure disease. stevekochscience.blogspot.com (2011).at <http://stevekochscience.blogspot.com/2011/03/i-am-maximally-skeptical-that-there.html> 15.Frauenfelder, H. et al. A unified model of protein dynamics. Proceedings of the National Academy of Sciences of the United States of America 106, 5129-34(2009). 16.Fenimore, P.W. et al. Bulk-solvent and hydration-shell fluctuations, similar to alpha- and beta-fluctuations in glasses, control protein motions and functions. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101, 14408-14413(2004). 17.Sidorova, N. & Rau, D. The dissociation rate of the EcoRI-DNA-specific complex is linked to water activity. Biopolymers 53, 363-368(2000). 18.Sidorova, N. Linkage of ecori dissociation from its specific dna recognition site to water activity, salt concentration, and ph: separating their roles in specific and non-Specific binding. Journal of Molecular Biology 310, 801-816(2001). 19.Koch, S.J. et al. Probing Protein-DNA Interactions by Unzipping a Single DNA Double Helix. Biophysical Journal 83, 1098-1105(2002). 20.Jiang, J. et al. Detection of high-Affinity and sliding clamp modes for msh2-Msh6 by single-Molecule unzipping force analysis. Molecular Cell 20, 771-781(2005). 21.Johnson, D.S. et al. Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase. Cell 129, 1299-1309(2007). 22.Shundrovsky, A. et al. Probing swi/snf remodeling of the nucleosome by unzipping single dna molecules. Nature Structural & Molecular Biology 13, 549-554(2006). 23.Hall, M.A. et al. High-Resolution dynamic mapping of histone-Dna interactions in a nucleosome. Nature Structural & Molecular Biology 16, 124-129(2009). 24.Koch, S.J. & Wang, M.D. Dynamic force spectroscopy of protein-DNA interactions by unzipping DNA. Physical review letters 91, 028103(2003). 25.Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophysical journal 72, 1541-55(1997). 26.Evans, E. Probing the relation between force--lifetime--and chemistry in single molecular bonds. Annu Rev Biophys Biomol Struct 30, 105-128(2001). 27.Koch, S.J. Tus (biology). Wikipedia (2009).at <http://en.wikipedia.org/w/index.php?title=Tus_(biology)&oldid=323433198> 28.Neylon, C. et al. Replication Termination in Escherichia coli : Structure and Antihelicase Activity of the Tus-Ter Complex. Society 69, 501-526(2005). 29.Maloney, A. Experimental protocols for and studies of the effects of surface passivation and water isotopes on the gliding speed of microtubules propelled by kinesin-1. (2011).at <http://repository.unm.edu/handle/1928/12872> 30.Maloney, A., Herskowitz, L.J. & Koch, S.J. Effects of surface passivation on gliding motility assays. PloS one 6, e19522(2011). 31.Deutsch, J.M., Brunner, M.E. & Saxton, W.M. The mechanics of a microscopic mixer: microtubules and cytoplasmic streaming in Drosophila oocytes. 7(2011).at <http://arxiv.org/abs/1101.2225> 32.Smith, S., Cui, Y. & Bustamante, C. Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules. Science 271, 795-799(1996). 33.Williams, M.C., Rouzina, I. & McCauley, M.J. Peeling back the mystery of DNA overstretching. Proceedings of the National Academy of Sciences of the United States of America 106, 18047-8(2009). 34.Koch, S.J. et al. Probing protein-DNA interactions by unzipping a single DNA double helix. Biophysical journal 83, 1098-105(2002). 35.Hänggi, P. & Borkovec, M. Reaction-rate theory: fifty years after Kramers. Reviews of Modern Physics 62, 251-341(1990).

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