Environmental Molecular Sciences Laboratory Pacific Northwest Laboratory

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Remote COSY NMR experiment for structure and
sequence determination of simple dipeptides
Anna G. Cavinato and Ronald B. Kelley,
Department of Chemistry and Biochemistry, Eastern
Oregon University, La Grande, OR 97850 229th
ACS Spring National Meeting, San Diego, CA March
16, 2005
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Outline

• Motivation
• The EMSL/EOU Collaboratory project
• Remote experiments
• Tools
• 2-D gCOSY NMR experiment
• Other correlation experiments
• Qualitative Assessment

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Eastern Oregon University is located 120 miles
south-east of Richland
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Some Facts about Eastern

• Founded in 1929
• Enrollment 2,000
• One of the seven public institutions in Oregon
• New 100,000 square feet science center

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The EMSL
Mission
. . . provide the fundamental scientific basis
needed to solve the nations environmental
problems.
. . . advance molecular science in support of the
long-term missions of the U.S. Department of
Energy.
National Scientific User Facility
. . . make unique research resources available to
DOE scientists and researchers from academia and
industry.
. . . provide opportunities needed to educate and
recruit young scientists to meet the demanding
environmental challenges of the future.
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Advanced Research Capabilities for Environmental
Molecular Science
Chambers For
Cluster
Molecular Level
Real Time
Controlled
Structure
Surface
Dynamics
Simulation Of
and Dynamics
Chemistry
Facility
Environmental
Facility
Reactions
Visualization
and
Ultrahigh
Graphics
Field NMR
Laboratory
Large Molecule
High
Mass
Performance
Spectrometry
Computing
Facility
Sensor, Design
Materials
Advanced
Interphase
Synthesis
Processing
Chemistry
Fabrication and
Testing
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EMSL Virtual NMR Facility

• High-field NMR capabilities
• Secure remote spectrometer control
• Desktop Conferencing
• Electronic Laboratory Notebook

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How to Get Started

• Identify a scientist collaborator at the VNMRF
• Submit a proposal to the EMSL NMR facility
• Download EMSL Collaboratory tools
• (http//collaboratory.emsl.pnl.gov)

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Hardware and Network Requirements

• Computer running Unix or windows
• Internet connection
• Camera
• Microphone (or speaker phone)

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Using VNC for Remote Access to the Spectrometer
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VNC Requirements

• Obtain appropriate computer account
• Obtain a secure shell client (ssh)
• Obtain the latest Netscape or Internet Explorer
  browsers to run a dynamic VNC applet.

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EMSL Collaborative Environment
Electronic Notebook
CORE2000 (real-time)
Audio/Video Teleconferencing
Multi White Board
Shared Window
Chat Box
Remote Instrument
And Analysis
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White Board
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The Electronic Notebook

• WWW based access to EMSL data
• Automation of data input from instruments and
  calculations
• Querying/ Searching
• Interactive input of results into notebook from
  the WWW
• Rich media types (text, images, files, 3D
  structures, voice, animations, video, ...)

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2-D gCOSY NMR experiment

• Determine structure and sequence of simple
  dipeptides
• Known Ala-Ser and Thr-Arg
• Unknown Thr-Asp

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Purpose of the experiment

• Expose students to state of the art
  instrumentation and real world problems.
• Simulate how structural biologists take advantage
  of 1-D and 2-D NMR to solve complex structures.
• Provide interaction with EMSL scientists for
  future research opportunities.

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Purpose of the experiment (continued)

• Assess technology-based instruction.
• Improve my personal knowledge and skills.
• Connect with scientists at a large research
  facility.

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EOU Chem 422 - Live from the EMSL
Remote 2-D COSY NMR Experiment
EOU - Oregon
Scientist David Hoyt - EMSL
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EOU Chem 422 - Live from the EMSL
Remote 2-D COSY NMR Experiment
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EOU Chem 422 - Live from the EMSL
Remote 2-D COSY NMR Experiment
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EOU Chem 422 - Live from the EMSL
Remote 2-D COSY NMR Experiment
2D COSY
1D spectrum
Ala-Ser spectra
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EOU Chem 422 - Live from the EMSL
Remote 2-D COSY NMR Experiment
Thr-Arg Spectra
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EOU Chem 422 - Live from the EMSL
Remote 2-D COSY NMR Experiment
Thr-Asp Spectra
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Supporting information
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Additional NMR experiment

• JEOL ECX class spectrometer operating at 400 MHz
• Autotune Field gradient probe. This probe does
  not require human intervention to change between
  nuclei making it easy for observing different
  nuclei from a remote site.
• 24 Auto sample changer
• Remote access directly over the Internet. No
  special software required.
• All experiments were conducted using standard
  automation provided by JEOL
• Samples were 5(w/v) in D2O
• Known Ala-Ser and Thr-Asp
• Unknown Asp-Ala

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Additional NMR experiments

• 1H
• 13C
• 2-D Gradient Assisted Double Quantum Filtered
  COSY (g-DQF-COSY) for homonuclear correlations
• Heteronuclear Correlation through Multiple
  Quantum Coherence (HMQC ) for 1H - 13C direct
  correlation
• Gradient Assisted Hetero Multiple Bond
  Correlation (g-HMBC) for 1H - 13C through bond
  long range correlation.

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Additional NMR experiments
Phe-ala-g-COSY
Phe-ala-1D
Phe-ala-HMQC
Phe-ala-HMBC
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Additional NMR experiments
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Remote ExperimentWorse, Better,

• Its not completely hands-on
• Lack of non-verbal cues
• Lack of presence
• Intrusion of Technology
• Delays, network outages
• Advanced computer skills

• Accessing instruments and resources that
  otherwise would be unavailable
• Student laboratory experience enhancement
• Faculty skills enhancement
• Student research enhancement

or Different?
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Acknowledgments

• The research was performed at the Environmental
  Molecular Science Laboratory (a national
  scientific user facility sponsored by the DOE
  Biological and Environmental Research) located at
  Pacific Northwest National Laboratory and
  operated for DOE by Battelle.
• Dr. David Hoyt, PNNL
• Mr. Michael Peterson, PNNL
• Dr. Ashok Krishnaswami, JEOL
• My students