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

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

3. Introduction Number of protein folds Number of protein folds Computational methods for identifying amino acid sequences compatible with target structure – not for protein creation Computational methods for identifying amino acid sequences compatible with target structure – not for protein creation Side Chains as Templates Side Chains as Templates New protein design => more rigorous test of current force fields and optimization methodology than redesign of naturally occurring proteins 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 Search of nearby conformational space and sequence space 2 methods of protein redesign (variation of backbone conformation and amino acid sequence) 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 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 Result: 93 residue protein with topology not in PDB

5. Structure to Sequence RosettaDesign predicts a amino acid sequence from a desired structure. RosettaDesign predicts a amino acid sequence from a desired structure. Input and Output Input and Output Input a structure you would like to create Input a structure you would like to create Output a amino acid sequence that will produce the structure with low free energy 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. RosettaDesign is used to predict protein structure from protein residue sequence. Input and Output Input and Output Input Amino Acid sequence Input Amino Acid sequence Output a Predicted near minimum free energy structure Output a Predicted near minimum free energy structure

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

8. Stability Thermally stable Thermally stable CD Spectrum at 98˚C is nearly indistinguishable from that at 25˚C 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 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 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 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 Crystallization Top7 yielded crystals that diffracted to 2.5 Å Top7 yielded crystals that diffracted to 2.5 Å Strong molecular replacement (MR) solution to phase problem Strong molecular replacement (MR) solution to phase problem This suggest design model very close to true structure 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 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 o A) in de novo Protein Design Atomic Level Accuracy (RMSD = 1.17 o A) in de novo Protein Design Validation of Accuracy of Potential Functions Validation of Accuracy of Potential Functions

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

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