This is the full Project Description for my 2011 NSF CAREER proposal. As I described on my blog, I am disappointed in the unfinished product, mostly because I still think the proposed research is important, exciting, and achievable by my lab. ( http://stevekochresearch.blogspot.com/2011/08/2011-nsf-career-proposal-ugh-failures.html )
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
How to do quick user assign in kanban in Odoo 17 ERP
2011 NSF CAREER_Steve Koch Full Project Description
1.
2. [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.
3. 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.
4. 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.<br />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.<br />Sub Aim 1.2: Determine whether growth rates of unicellular organisms are slowed in DDW.<br />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. <br />Specific Aim 2: Gain atomistic understanding of water isotope effects and osmotic stress on biomolecule stability and biomolecular interactions <br />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. <br />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.<br />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. <br />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. <br />V. Educational Integration and Broader Impacts<br />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. <br />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.<br />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.<br />Integration of research with university education. <br />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).<br />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.<br />References Cited<br />Mendeley Bibliography1.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 />3.Bradley, J.-C. et al. Collaboration Using Open Notebook Science in Academia. Collaborative Computational Technologies for Biomedical Research (2011).<br />4.Lewis, G.N. The biochemistry of water containing hydrogen isotope. Journal of the American Chemical Society 55, 3503–3504(1933).<br />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).<br />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).<br />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).<br />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).<br />9.Chakrabarti, G. et al. Stabilization of tubulin by deuterium oxide. Biochemistry 38, 3067-72(1999).<br />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).<br />11.Somlyai, G. et al. Naturally occurring deuterium is essential for the normal growth rate of cells. FEBS letters 317, 1-4(1993).<br />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).<br />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).<br />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><br />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).<br />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).<br />17.Sidorova, N. & Rau, D. The dissociation rate of the EcoRI-DNA-specific complex is linked to water activity. Biopolymers 53, 363-368(2000).<br />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).<br />19.Koch, S.J. et al. Probing Protein-DNA Interactions by Unzipping a Single DNA Double Helix. Biophysical Journal 83, 1098-1105(2002).<br />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).<br />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).<br />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).<br />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).<br />24.Koch, S.J. & Wang, M.D. Dynamic force spectroscopy of protein-DNA interactions by unzipping DNA. Physical review letters 91, 028103(2003).<br />25.Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophysical journal 72, 1541-55(1997).<br />26.Evans, E. Probing the relation between force--lifetime--and chemistry in single molecular bonds. Annu Rev Biophys Biomol Struct 30, 105-128(2001).<br />27.Koch, S.J. Tus (biology). Wikipedia (2009).at <http://en.wikipedia.org/w/index.php?title=Tus_(biology)&oldid=323433198><br />28.Neylon, C. et al. Replication Termination in Escherichia coli : Structure and Antihelicase Activity of the Tus-Ter Complex. Society 69, 501-526(2005).<br />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><br />30.Maloney, A., Herskowitz, L.J. & Koch, S.J. Effects of surface passivation on gliding motility assays. PloS one 6, e19522(2011).<br />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><br />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).<br />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).<br />34.Koch, S.J. et al. Probing protein-DNA interactions by unzipping a single DNA double helix. Biophysical journal 83, 1098-105(2002).<br />35.Hänggi, P. & Borkovec, M. Reaction-rate theory: fifty years after Kramers. Reviews of Modern Physics 62, 251-341(1990). <br />