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Methods and Results

Methods and Results. Darius Hines Kyle Priver Science Research Connections Summer 2010. What’s this about?. The enzyme dihydrofolate reductase (DHFR) is found everywhere and is a vital component in the productions of purines.

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Methods and Results

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  1. Methods and Results Darius Hines Kyle Priver Science Research Connections Summer 2010

  2. What’s this about? • The enzyme dihydrofolate reductase (DHFR) is found everywhere and is a vital component in the productions of purines. • Development of accurate, useful computer models of this enzyme is critical to further research into the function of this molecule. • Of specific interest is the potential for using DHFR models to investigate hydrogen tunneling during enzyme reactions.

  3. Methods Overview • Acquire protein database files for models of the enzyme with methotrexate at the active site and the folate molecule • Replace the methotrexate with the folate molecule

  4. Methods Overview • Obtain a protein structure file for the modified enzyme model • Run computer simulations to analyze the modified enzyme model for stability

  5. Methotrexate coordinates provide best match to enzyme active site DHFR from E. coli treated with methotrexate is source Folate molecule located online in file library Acquisition of Protein Database Files Enzyme with Methotrexate Folate Molecule

  6. Replace Methotrexate with Folate • The Visual Molecular Dynamics program is used to align DHFR of E. coli with DHFR of Thermatoma maritima • E coli is deleted, leaving methotrexate at Thermatoma maritima DHFR acitve site

  7. Align the Folate molecule with the methotrexate Delete the methotrexate, saving coordinates. Replace Methotrexate with Folate

  8. Obtain a protein structure file for the modified enzyme Use the Nanoscale Molecular Dynamics program to generate a file that contains all information necessary to apply a force field to the modified enzyme

  9. Does the overall molecule stay together or fly apart? Does the folate molecule stay at the active site or move away? Run simulations to analyze the modified enzyme for stability

  10. Does the molecule fly apart? Root Mean Square Deviation for Aligned Folate – Entire Enzyme Although the lines seem to possess a degree of periodicity at three different investigated temperatures, the range of the fluctuations seems to be constant. The middle temperature seems to have the largest range of fluctuation. RMSD (Å) Time (ps)

  11. Does the folate remain at the active site? Root Mean Square Deviation for Aligned Folate – Active Site There seems to be a leveling off at the lowest investigated temperature while the folate seems to have more motion a the highest temperature RMSD (Å) Time (ps)

  12. Results and Next Steps • The model seems to be more stable at the lower temperatures, possibly because at the higher temperatures, uneven distributions of energy produce more pronounced localized motions. • The folate seemed to be more mobile at the mid temperature, perhaps because of some resonance effect between the molecular structure and the frequency of motion imparted by that particular level of energy. • Run more simulations to verify consistency of results. • Use model of modified enzyme to further investigation of hydrogen tunneling during enzyme reactions.

  13. The End!

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