This is the first section of my 2011 NSF CAREER proposal. As of August 1, 2011, I haven't published the remaining sections yet, but will link when I do. Here are links to prior years' proposals, which were declined: * 2009 http://www.scribd.com/doc/17548381/2009-ProposalCAREER-SingleMolecule-Analysis-of-Genomic-DNA-and-Chromatin-in-Eukaryotic-Transcription * 2008 http://www.scribd.com/doc/10196076/2008-NSF-CAREERproposal-Only

1. I. Specific Aims<br />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 formsADDIN Mendeley Citation{b2df1b3e-26a2-102d-8183-0024e85e2bb9} CSL_CITATION { quot;
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<m:note>Interesting thoughts from Lewis in his 1934 Science paper on biological effects of heavy water (relevant to the FEBS paper I read a couple weeks ago http://friendfeed.com/stevekoch/a11da170/naturally-occurring-deuterium-is-essential): 0026quot;It is not inconceivablethat heavy hydrogen, which exists in small amountsin all natural water, may actually be essential to someplants or animals. A supply of water almost completelyfreed from the heavy isotope is now beingprepared for the purpose of conducting such studies.0026quot;<m:linebreak/> <m:linebreak/> </m:note>quot;
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THE BIOLOGY OF HEAVY WATER.quot;
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} 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. <br />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 1934ADDIN Mendeley Citation{b2df1b3e-26a2-102d-8183-0024e85e2bb9} CSL_CITATION { quot;
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<m:note>Interesting thoughts from Lewis in his 1934 Science paper on biological effects of heavy water (relevant to the FEBS paper I read a couple weeks ago http://friendfeed.com/stevekoch/a11da170/naturally-occurring-deuterium-is-essential): 0026quot;It is not inconceivablethat heavy hydrogen, which exists in small amountsin all natural water, may actually be essential to someplants or animals. A supply of water almost completelyfreed from the heavy isotope is now beingprepared for the purpose of conducting such studies.0026quot;<m:linebreak/> <m:linebreak/> </m:note>quot;
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} 1 and remains untested! We will test it using plant seeds, E. coli, and yeast. The two sub-aims are:<br />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. <br />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.<br />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).<br />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.<br />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. <br />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.<br />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.<br />In 1995, Parsegian and colleagues stressed that the fundamental importance of water activity is so often overlooked in molecular biologyADDIN Mendeley Citation{7958f0f4-a550-4ae0-983e-697ded46f8ab} CSL_CITATION { quot;
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} 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.<br />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. <br />Mendeley Bibliography CSL_BIBLIOGRAPHY 1.Lewis, G.N. THE BIOLOGY OF HEAVY WATER. Science (New York, N.Y.) 79, 151-153 (1934).<br />2.Parsegian, V.A., Rand, R.P. & Rau, D.C. Macromolecules and water: probing with osmotic stress. Methods in enzymology 259, (1995). <br />