Talk given by Steve at the Unviversity of New Mexico Chemistry Department on September 11, 2009. It is mostly still an introduction to our kinesin project, but now I'm able to include the latest results from gliding motility assay, tracking software, and stochastic kinetics simulation.
Probing Molecular Electronic Structure Using High Harmonic Generation TomographyChelsey Crosse
The structure of valence electronic orbitals of a molecule determines the majority of chemical properties. Generation of high-order harmonic frequencies from atomic sources has been directly related to the electronic structure of the atom, (1) and extended as far as tomographic reconstruction of linearly symmetric polyatomic molecular systems with some success. (2,3,4)
However, because of the increased resolution of these reconstructions, discrimination of fine details of the orbital reconstructions reveals some inconsistencies in the orbital shapes when compared with past models & theoretical calculations. (2) There are several proposed corrections to the Strong Field Approximation (SFA) that currently underlies tomographic reconstruction as well as all other experiments that use high harmonic generation (HHG) to probe molecular systems. (5,6,7)
---------------------------------------------------------------------
1. Lewenstein et al. Phys Rev A 49 (3) 1994.
2. Salieres, Maquet, Haessler, Caillat, Taieb. Rep. Prog. Phys. 75 (2012) 062401.
3. Li, Liu, Yang, Song, Zhao, Lu, Li, Xu. Opt. Ex. 21 (6) 2013. 7599.
4. Torres et al. Phys Rev. Lett. 98 (2007) 203007.
5. Diveki et. al. J. Chem Phys. 414 (2013) 121.
6. Yip, Palacios, Rescigno, McCurdy, Martin. J. Chem Phys 414 (2013) 112.
7. Spanner, Patchkovskii. J. Chem. Phys. 414 (2013) 10.
Join Brian Searle on an illustrated tour about interpreting MS/MS peptide spectra. On this tour you will first see how you can relate mass spectra to peptides. Next you see why the SEQUEST software was developed to interpret these spectra as peptides. Next you will see other software approaches have been developed and how combining approaches produces even better results.
brief but informative knowledge about what basically PET is and what is the phenomenon behind this machine ... easy to understand as well as presenting during lectures and in classes . share it
Dr. AVS Suresh, MD, DM, ECMO, Consultant Hemato-Oncologist, Chief Scientific Officer & Director, ClinSync, on the man-made as well as other kind of EMF radiation.
Probing Molecular Electronic Structure Using High Harmonic Generation TomographyChelsey Crosse
The structure of valence electronic orbitals of a molecule determines the majority of chemical properties. Generation of high-order harmonic frequencies from atomic sources has been directly related to the electronic structure of the atom, (1) and extended as far as tomographic reconstruction of linearly symmetric polyatomic molecular systems with some success. (2,3,4)
However, because of the increased resolution of these reconstructions, discrimination of fine details of the orbital reconstructions reveals some inconsistencies in the orbital shapes when compared with past models & theoretical calculations. (2) There are several proposed corrections to the Strong Field Approximation (SFA) that currently underlies tomographic reconstruction as well as all other experiments that use high harmonic generation (HHG) to probe molecular systems. (5,6,7)
---------------------------------------------------------------------
1. Lewenstein et al. Phys Rev A 49 (3) 1994.
2. Salieres, Maquet, Haessler, Caillat, Taieb. Rep. Prog. Phys. 75 (2012) 062401.
3. Li, Liu, Yang, Song, Zhao, Lu, Li, Xu. Opt. Ex. 21 (6) 2013. 7599.
4. Torres et al. Phys Rev. Lett. 98 (2007) 203007.
5. Diveki et. al. J. Chem Phys. 414 (2013) 121.
6. Yip, Palacios, Rescigno, McCurdy, Martin. J. Chem Phys 414 (2013) 112.
7. Spanner, Patchkovskii. J. Chem. Phys. 414 (2013) 10.
Join Brian Searle on an illustrated tour about interpreting MS/MS peptide spectra. On this tour you will first see how you can relate mass spectra to peptides. Next you see why the SEQUEST software was developed to interpret these spectra as peptides. Next you will see other software approaches have been developed and how combining approaches produces even better results.
brief but informative knowledge about what basically PET is and what is the phenomenon behind this machine ... easy to understand as well as presenting during lectures and in classes . share it
Dr. AVS Suresh, MD, DM, ECMO, Consultant Hemato-Oncologist, Chief Scientific Officer & Director, ClinSync, on the man-made as well as other kind of EMF radiation.
Osmotic stress and water isotope effects in kinesin-1 gliding motility assaysSteve Koch
The osmotic pressure and kinetic properties of water play important roles in biomolecular interactions. As pointed out by Parsegian, Rand, and Rau, these crucial roles are often overlooked1. In some fields, osmotic stress and isotope effects have been exploited for probing the role water plays in binding interactions of biomolecules. To our knowledge, there have been no studies of osmotic stress and water isotope effects for kinesin, and only a handful for myosin. We’re currently using the gliding motility assay to see whether we can extract new information about kinesin-1 / microtubule interactions by changing osmotic stress and water isotopes. We will describe our open-source, automated analysis platform for extracting microtubule gliding speeds from image series. We will also show our preliminary analyses of the changes seen in gliding assays when done in heavy water (either heavy-hydrogen or heavy-oxygen) or osmolytes (betaine). We will discuss whether osmotic stress and isotopes, particularly heavy-oxygen water, might be an important tool for probing effects of water on binding interactions between kinesin and microtubules. We will also discuss potential applications of deuterium water for stabilizing microtubules and kinesin for lab or device applications.
[1] Parsegian, V. A., Rand, R. P., & Rau, D. C. (1995). Macromolecules and water: probing with osmotic stress. Methods in Enzymology, 259.
This work was supported by the DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008 in collaboration with Dr. Susan Atlas lab (UNM).
Talk multiscale analysis of ionic solutions is unavoidableBob Eisenberg
Ions in channels and solutions control most living functions. Analysis in atomic detail is needed, but so is prediction of functions on the macroscopic scale. Computational electronics has solved similar issues and we all benefit from the computational devices it provides us. These slides show how a similar approach can be used, and is necessary in my view, for ions solutions and biological systems, most notably in ion channels
Device approach to biology and engineeringBob Eisenberg
Device Approach to Biology (and Engineering)
The goal of biological research is often more to control than to understand. Devices in biology (like ion channels) control individual functions just as they do in our technology. Study of control requires a multiscale approach because a handful of atoms, moving in 10-15 sec, control biological functions extending meters and taking seconds. Structural biology and molecular dynamics are essential (and beautiful!) parts of this hierarchy, but so are the functions themselves, and the electric field equations that link structure and function on all scales from atoms to nerve cells. Analyzing biological systems as devices is usually successful, and almost always productive.
Quantum entanglement is a phenomenon in theoretical physics that happens when pairs or groups of particles are generated in such a way that the quantum state of each particle cannot be described independently of the others, even when the particles are separated by a large distance. Instead, a quantum state must be described for the system as a whole. Based on the theory of cancer as an evolutionary metabolic disease (Evolutionary Metabolic Hypothesis of Cancer or EMHC), the cancerous cells are eukaryotic cells with different metabolic rate from healthy cells due to the damaged or shut down mitochondria in them. Assuming each human eukaryotic cell as a particle and the whole body as a Quantum Entangled System (QES), is a new perspective on the description of cancer disease, and this link between theoretical physics and biological sciences in the field of cancer therapies can be a new insight into the cause, prevention and treatment of cancer. Additionally, this perspective admits the Lamarckian evolution in the understanding of the mentioned disease. We have presented each human eukaryotic cell containing mitochondria as a QES, and the whole body containing healthy and normal cells as a QES as well. The difference between the entropy of the healthy cells and cancer cells has also been mentioned in this research.
Keywords: Quantum Entanglement, Cancer, Mitochondria, Evolution, Quantum Entangled System (QES), EMHC
An alternative to the "big molecules" view of proteins is the "small things" view in which protein have a shape and material properties. This talk is about investigating these properties.
The great basic question of science: Membrane compartment or non-membrane pha...Vladimir Matveev
Oral presentation at The 2nd All-Russian Conference on Astrobiology. Moscow, Pushchino, 5-9 June 2016.
Video in English: https://youtu.be/Hn7A-1w0tuQ
Presentation slides in English as pdf:
http://www.bioparadigma.spb.ru/conf/Matveev-2016-The.great.basic.question.of.science_Eng_Slides.pdf
Comments for slides in English:
http://www.bioparadigma.spb.ru/conf/Matveev-2016-The.great.basic.question.of.science_Comments_Eng.pdf
The Wonderful World of Scanning Electrochemical Microscopy (SECM)InsideScientific
To watch the webinar, go to:
https://insidescientific.com/webinar/the-wonderful-world-of-scanning-electrochemical-microscopy-secm/
In this webinar, Dr. Janine Mauzeroll discusses the fundamentals, critical experimental parameters and recent applications for scanning electrochemical Microscopy (SECM).
In its simplest form, SECM is a scanning probe technique in which a small-scale electrode is scanned across an immersed substrate while recording the current response. This response is dependent on both the surface topography and the electrochemical activity of the substrate. Consequently, using an array of operational modes, a wide variety of substrates and experimental systems can be characterized. The strength of SECM lies in its ability to quantify material flux from a surface with a high spatial and temporal resolution. It has been used in a variety of applications fields.
Dr. Janine Mauzeroll describes the fundamentals of SECM, including the required instrumentation and the principles of the most frequently used operational modes. Following this basic understanding of SECM principles, she then moves towards a comprehensive summary of the critical parameters for any SECM experiment. Specifically, she discusses in detail redox mediators, probes, and solvent systems that are used in SECM experiments. Finally, she presents recent applications of SECM with an emphasis on her work in the last five years related to material characterization, corrosion and batteries.
Lens as an Osmotic Pump, a Bidomain Model. DOI: 10.13140/RG.2.2.25046.80966Bob Eisenberg
The lens of the eye has no blood vessels to interfere with vision. The lens is far too large for diffusion to provide food and clear wastes.
Experimental, theoretical and computational work has shown that the lens supports its own microcirculation. It is an osmotic pump that implements what physiologists have long believed “Convection provides what diffusion cannot.”
We introduce a general (non-electro-neutral) model that describes the steady-state relationships among ion fluxes, water flow and electric field inside cells, and in the narrow extracellular spaces within the lens.
Using asymptotic analysis, we derive a simplified model exploiting the numerical values of physiological parameters. The model reduces to first generation ‘circuit’ models and shows the basis of computer simulations too large to easily understand. The full model helps resolve paradoxes that have perplexed molecular biologists: crucial physiological properties do not depend as expected on the permeability of the lens interior (to water flow).
Dr. Patrick Bradshaw presents an overview of his program, Human Performance and Biosystems, at the AFOSR 2013 Spring Review. At this review, Program Officers from AFOSR Technical Divisions will present briefings that highlight basic research programs beneficial to the Air Force.
2011 NSF CAREER_Steve Koch Full Project Description Steve Koch
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
This is a summary I gave at group meeting today on what I'd learned about D2O (aka "heavy water" aka "deuterium oxide") and its effects on biochemistry/biophysics of enzymes and proteins.
Here are the slides describing talents and hedgehog concepts in the context of students' future careers. It's the background to our final assignment for the semester: http://openwetware.org/wiki/User:Steven_J._Koch/Talents_assignment
Discussion of dispersion and rainbows. Also some cool photos of blackbody and fluorescent spectra from Tom Decaro and Analisa Goodman as part of the homework question.
08 Feb 17 Light, Electron E Levels Actual PresentedSteve Koch
Introduction to electromagnetic radiation and light. Viewing atomic spectra with diffraction gratings. Optical tweezers (cool example of light having momentum).
Epistemic Interaction - tuning interfaces to provide information for AI supportAlan Dix
Paper presented at SYNERGY workshop at AVI 2024, Genoa, Italy. 3rd June 2024
https://alandix.com/academic/papers/synergy2024-epistemic/
As machine learning integrates deeper into human-computer interactions, the concept of epistemic interaction emerges, aiming to refine these interactions to enhance system adaptability. This approach encourages minor, intentional adjustments in user behaviour to enrich the data available for system learning. This paper introduces epistemic interaction within the context of human-system communication, illustrating how deliberate interaction design can improve system understanding and adaptation. Through concrete examples, we demonstrate the potential of epistemic interaction to significantly advance human-computer interaction by leveraging intuitive human communication strategies to inform system design and functionality, offering a novel pathway for enriching user-system engagements.
PHP Frameworks: I want to break free (IPC Berlin 2024)Ralf Eggert
In this presentation, we examine the challenges and limitations of relying too heavily on PHP frameworks in web development. We discuss the history of PHP and its frameworks to understand how this dependence has evolved. The focus will be on providing concrete tips and strategies to reduce reliance on these frameworks, based on real-world examples and practical considerations. The goal is to equip developers with the skills and knowledge to create more flexible and future-proof web applications. We'll explore the importance of maintaining autonomy in a rapidly changing tech landscape and how to make informed decisions in PHP development.
This talk is aimed at encouraging a more independent approach to using PHP frameworks, moving towards a more flexible and future-proof approach to PHP development.
DevOps and Testing slides at DASA ConnectKari Kakkonen
My and Rik Marselis slides at 30.5.2024 DASA Connect conference. We discuss about what is testing, then what is agile testing and finally what is Testing in DevOps. Finally we had lovely workshop with the participants trying to find out different ways to think about quality and testing in different parts of the DevOps infinity loop.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
LF Energy Webinar: Electrical Grid Modelling and Simulation Through PowSyBl -...DanBrown980551
Do you want to learn how to model and simulate an electrical network from scratch in under an hour?
Then welcome to this PowSyBl workshop, hosted by Rte, the French Transmission System Operator (TSO)!
During the webinar, you will discover the PowSyBl ecosystem as well as handle and study an electrical network through an interactive Python notebook.
PowSyBl is an open source project hosted by LF Energy, which offers a comprehensive set of features for electrical grid modelling and simulation. Among other advanced features, PowSyBl provides:
- A fully editable and extendable library for grid component modelling;
- Visualization tools to display your network;
- Grid simulation tools, such as power flows, security analyses (with or without remedial actions) and sensitivity analyses;
The framework is mostly written in Java, with a Python binding so that Python developers can access PowSyBl functionalities as well.
What you will learn during the webinar:
- For beginners: discover PowSyBl's functionalities through a quick general presentation and the notebook, without needing any expert coding skills;
- For advanced developers: master the skills to efficiently apply PowSyBl functionalities to your real-world scenarios.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Slack (or Teams) Automation for Bonterra Impact Management (fka Social Soluti...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on the notifications, alerts, and approval requests using Slack for Bonterra Impact Management. The solutions covered in this webinar can also be deployed for Microsoft Teams.
Interested in deploying notification automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
Search and Society: Reimagining Information Access for Radical FuturesBhaskar Mitra
The field of Information retrieval (IR) is currently undergoing a transformative shift, at least partly due to the emerging applications of generative AI to information access. In this talk, we will deliberate on the sociotechnical implications of generative AI for information access. We will argue that there is both a critical necessity and an exciting opportunity for the IR community to re-center our research agendas on societal needs while dismantling the artificial separation between the work on fairness, accountability, transparency, and ethics in IR and the rest of IR research. Instead of adopting a reactionary strategy of trying to mitigate potential social harms from emerging technologies, the community should aim to proactively set the research agenda for the kinds of systems we should build inspired by diverse explicitly stated sociotechnical imaginaries. The sociotechnical imaginaries that underpin the design and development of information access technologies needs to be explicitly articulated, and we need to develop theories of change in context of these diverse perspectives. Our guiding future imaginaries must be informed by other academic fields, such as democratic theory and critical theory, and should be co-developed with social science scholars, legal scholars, civil rights and social justice activists, and artists, among others.
"Impact of front-end architecture on development cost", Viktor TurskyiFwdays
I have heard many times that architecture is not important for the front-end. Also, many times I have seen how developers implement features on the front-end just following the standard rules for a framework and think that this is enough to successfully launch the project, and then the project fails. How to prevent this and what approach to choose? I have launched dozens of complex projects and during the talk we will analyze which approaches have worked for me and which have not.
FIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdf
2009 September Kinesin Talk at UNM Chemistry
1. Biophysical Studies of the Molecular Motor Kinesin Koch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM) Steve Koch, DTRA Co-PI, Experimental Lead Asst. Prof. Physics and Astronomy Larry Herskowitz, IGERT Fellow Physics Ph.D. Student Anthony Salvagno, IGERT Fellow Physics Ph.D. Student Brigette Black Physics Ph.D. Student Andy Maloney, NSF IGERT Fellow Physics Ph.D. Student Igor Kuznetsov Postdoc Linh Le Physics B.S. @ UNM Now biophysics grad @ Ohio State “ Kiney” Brian Josey Physics B.S. Student This talk was presented at the UNM department of chemistry on Sept. 11, 2009. I have tried to give appropriate credit on all images used—Steve Koch
2.
3.
4. Kinesin is a eukaryotic molecular motor protein with a number of intracellular functions Mitosis Intracellular transport
5. Kinesin binds to microtubules and uses ATP hydolysis to walk along tubulin protofilaments An overview of the two basic components of this system: Microtubules Kinesin Microtubules are a key component of the system: kinesin does not move or catalyze ATP hydrolysis in absence of MTs Goldstein Lab
7. Microtubules can be reliably polymerized in vitro In living cells, predominant form of MTs have 13 protofilaments (PFs) In vitro “reassembly” of microtubules was possible by the early 1970s (Borisy, Brinkley, …) Typically performed with purified bovine or porcine brain tubulin Produces an assortment of MTs with varying numbers of PFs (usually not 13) Recombinant tubulin is not readily available MTs are stabilized by taxol … chemical cross-linking is another strategy Easily visualized by fluorescence microscopy - + tubulin dimer Protofilament 25 nm
8. Kinesin binds to microtubules and uses ATP hydrolysis to walk along tubulin protofilaments An overview of the two basic components of this system: Microtubules Kinesin Goldstein Lab
9. Kinesin is a eukaryotic molecular motor protein with a number of intracellular functions Mitosis Intracellular transport Vale, Reese, Sheetz, 1985, Cell 42 39-50. “Identification of a Novel Force-Generating Protein, Kinesin, Involved in Microtubule-Based Motility.” At least 14 families of kinesin across all eukaryotes Dimeric “conventional” kinesin-1 : vesicle transport Kinesin-1, -2, -3, etc… E.g., Kinesin-5 is tetrameric kinesin: spindle formation HHMI Winter Bulletin 2005 Kinesin-5 tetramers
13. Truncated, tagged conventional kinesin constructs Coy, Hancock, Wagenbock, Howard (1999) Full length conventional kinesin self-inhibits by tail binding to motor domain Asbury, Fehr, Block (2003) Recombinant kinesin expressed in E. coli, purified by his-tag methods Limited commercial availability
14. Striving for atomistic insights into catalytic mechanism Sablin and Fletterick, 2004 JBC Much has been learned about kinesin at the stochastic (mechanical) level But atomistic understanding of mechanochemistry is lacking Our goal is to gain atomistic insight via a variety of experiments and simulations
15. Our DTRA Project: “Coupled Atomistic Modeling and Experimental Studies of Energy Transduction and Catalysis in the Molecular Motor Protein Kinesin” Susan Atlas and Steve Valone (LANL) “ Charge transfer embedded atom model” (CT-EAM) Atomistic modeling of kinesin catalytic core Kochlab: Biophysical studies of kinesin
16. An initial connection between theory & experiment: Water! CT-EAM can correctly model water; can make predictions about how osmotic pressure and heavy water will affect kinetics Kochlab: Can vary water osmotic pressure; heavy / light water
18. The osmotic stress method relies on changing water activity by adding high concentration of solutes Parsegian, Rand, Rau, Methods in Enzymology 259 (1995) “ Osmolyte” (sucrose, betaine, PEG, …) Reduces the chemical potential of water Molecule of interest has a shell of hydrating water molecules
19. No osmotic stress studies of kinesin untapped Utility proven in protein-DNA studies Protein DNA Non-specific, K nonsp Specific complex, K sp Sidorova and Rau, PNAS 1996
20. Osmotic stress dramatically increases lifetime of bound molecular complexes Osmotic pressure helpful For increasing lifetime too ln(Fraction bound) Sidorova and Rau Kinesin binding / unbinding
21. Why is water so important? Each time the kinesin head binds to tubulin, dozens of “hydrating” water molecules must be excluded. Each time the kinesin unbinds, water must “rehydrate” Thus, “water activity” strongly impacts binding kinetics (and whole kinetic cycle) Okada, Higuchi, Hirokawa Water excluded Water hydrating
22. Osmotic stress increases myosin-actin affinity (only one study I’m aware of) Highsmith et al. Biophys. J. 1996 No data exist for kinesin-MT Potentially many high-impact results
23. So, our first line of experiments will utilize osmotic stress (and heavy water) Properties of water will provide initial strong ties between theory and experiment Provide a very interesting line of high-impact experiments Also provide a connection to technological applications of kinesin / MT system Long-term stability of kinesin and microtubules Up-modulation of kinesin processivity? velocity? strength?
24. We will utilize two independent experimental platforms “ Easy” Robust Many experimental “knobs” Limited readout More difficult Many experimental “knobs” Many readout variables
25. Gliding motility assay Andy, Brigette, Linh have gliding assay working very well in our lab! Passivated glass surface (casein) Buffer includes ATP, antifade cocktail The assay is a bit finicky…light-induced microtubule disintegration is one problem.
26. Microtubule velocity in gliding assay is measured via LabVIEW image tracking software written by Larry
27. Gliding motility assay will be our initial main assay Operate in the high motor density regime Main experimental result is transport velocity Osmotic stress Light / heavy water Temperature, metal ions, ATP concentration Site-directed mutagenesis Experimental “knobs” to obtain data that can be compared with theory in the iterative loop Passivated glass surface (casein) Buffer includes ATP, antifade cocktail
28. Bead motility assay Adrian Fehr, Chip Asbury Science 2003 Steve Block Lab, Stanford Single-molecule kinesin transport Steve Block Lab, Stanford
29. Optical tweezers are formed by shining laser light into a high numerical aperture objective Optical Trap “ Laser tweezers” Microsphere Biomolecular “Tether” Coverglass Kochlab Optical Tweezers
30.
31.
32. Bead motility assay provides wealth of information High kinesin concentration Measure velocity of collective molecular motors (similar to gliding assay) Low kinesin concentration Single-molecule studies of kinesin: processivity force-velocity pull-off force Block et al. (2003) PNAS
33. Bead motility assay High kinesin concentration Measure velocity of collective molecular motors (similar to gliding assay) Low kinesin concentration Single-molecule studies of kinesin: processivity force-velocity pull-off force Experimental knobs for iterative theory/experiment loop: Osmotic stress Light / heavy water Temperature, metal ions, ATP concentration Site-directed mutagenesis We’ve not yet implemented this assay
34. Modeling: Kinesin kinetic cycle is complicated Gilbert et al., Nature 1995 R. D. Vale and R. A. Milligan, Science 288 , 88 (2000). We can not measure all of these rate constants directly…We measure: velocity; processivity; stall force; pull-off force
35. We have written a stochastic simulation for interpreting / predicting results of assays DT Gillespie , “ Exact Stochastic Simulation of Coupled Chemical Reactions” The Journal of Physical Chemistry, V. 8 p. 2340 1977 State machine, written in LabVIEW by Larry
36. Results of simulation can be viewed with LabVIEW animation – gives insight into kinetic pathway
38. Repeating many times, we can measure the “processivity” -> average run length Guydosh @ Block Nature 2009 optical tweezers measurements
39. Changing ATP concentration in the simulation produces Michaelis-Menten kinetics 200 simulations at each concentration
40. Application of opposing and assisting force also reproduces results seen with OT data! Block et al. (2003) PNAS
41. Acknowledgments Susan Atlas —Lead of the DTRA project UNM Physics / Cancer Center / Director of CARC Steve Valone —Co-PI (LANL) Haiqing Liu —Microdevice applications of kinesin LANL & Center for Integrated Nanotechnology (CINT) Evan Evans Lab —Single-molecule thermodynamics and kinetics U. New Mexico / U. British Columbia / Boston U. Collaborations Funding DTRA —Basic Science; CHTM —Startup; ACS —Jan Oliver IRG Our Lab —Larry Herskowitz, Andy Maloney, Brigette Black, Anthony Salvagno, Linh Le, Brian Josey, Igor Kuznetzov
43. Our preliminary data showed that osmotic stress effects protein-DNA unbinding forces X-intercept of these curves reveals off-rate Evans & Ritchie 1997 theory Protein-DNA interactions probed by DNA unzipping is another Koch Lab project We anticipate similar effects of osmotic stress on kinesin-MT forced disruption Specific Non-specific
44. Kinesin-microtubule unbinding forces Kawaguchi, Uemura, Ishiwata 2003 “ Dynamic Strength of Molecular Adhesion Bonds” Evan Evans and Ken Ritchie, 1997 Biophys. J. Brower-Toland et al., 2002