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de novo Protein Design

de novo Protein Design. Presented by Alison Fraser, Christine Lee, Pradhuman Jhala, Corban Rivera. Outline. Introduction Computational methods used for sequence and structure Biophysical and structural characteristics of novel protein Conclusion. Introduction. Number of protein folds

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de novo Protein Design

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  1. de novo Protein Design Presented by Alison Fraser, Christine Lee, Pradhuman Jhala, Corban Rivera

  2. Outline • Introduction • Computational methods used for sequence and structure • Biophysical and structural characteristics of novel protein • Conclusion

  3. Introduction • Number of protein folds • Computational methods for identifying amino acid sequences compatible with target structure – not for protein creation • Side Chains as Templates • New protein design => more rigorous test of current force fields and optimization methodology than redesign of naturally occurring proteins

  4. Introduction (continued) • Search of nearby conformational space and sequence space • 2 methods of protein redesign (variation of backbone conformation and amino acid sequence) • Development of procedure for identifying low free energy sequence-structure pairs that iterates between sequence optimization and structure prediction • Result: 93 residue protein with topology not in PDB

  5. Structure to Sequence • RosettaDesign predicts a amino acid sequence from a desired structure. • Input and Output • Input a structure you would like to create • Output a amino acid sequence that will produce the structure with low free energy

  6. Sequence to Structure • RosettaDesign is used to predict protein structure from protein residue sequence. • Input and Output • Input Amino Acid sequence • Output a Predicted near minimum free energy structure

  7. How does Top7 compare to proteins in nature? • Folding • Stability • Structure

  8. Stability • Thermally stable • CD Spectrum at 98˚C is nearly indistinguishable from that at 25˚C

  9. Folding of Top7 • At intermediate concentrations (~5 M) of guanidine hydrochloride (GuHCl) Top7 unfolds cooperatively • Steep transition in chemical denaturation is characteristic of the two-state unfolding expected for small, two-state, monomeric single-domain protein

  10. Structure • Nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum coherence (HSQC) exhibit features characteristic of protein with substantial beta-sheet content

  11. Comparing Top7 to model • Crystallization • Top7 yielded crystals that diffracted to 2.5 Å • Strong molecular replacement (MR) solution to phase problem • This suggest design model very close to true structure • Top7 crystal like the model was also judged to be a novel topology by TOPS server

  12. Comparison of model (blue) and the solved x-ray structure (red)

  13. Implications • Atomic Level Accuracy (RMSD = 1.17 oA) in de novo Protein Design • Validation of Accuracy of Potential Functions

  14. Reasons • Optimization of Sequence and Structure • No Functional Constraints • Extensive Optimization • No kinetics

  15. Possible Future Impacts • Synthetic proteins • Protein Therapeutics and Molecular Mechanics

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