de novo protein design
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
de novo Protein Designde novo Protein Design
Presented byPresented by
Alison Fraser, Christine Lee, Alison Fraser, Christine Lee, Pradhuman Jhala, Corban Pradhuman Jhala, Corban
RiveraRivera
OutlineOutline
IntroductionIntroduction Computational methods used for Computational methods used for
sequence and structuresequence and structure Biophysical and structural Biophysical and structural
characteristics of novel proteincharacteristics of novel protein ConclusionConclusion
IntroductionIntroduction Number of protein foldsNumber of protein folds Computational methods for identifying Computational methods for identifying
amino acid sequences compatible with amino acid sequences compatible with target structure – not for protein target structure – not for protein creationcreation
Side Chains as TemplatesSide Chains as Templates New protein design => more rigorous New protein design => more rigorous
test of current force fields and test of current force fields and optimization methodology than optimization methodology than redesign of naturally occurring proteinsredesign of naturally occurring proteins
Introduction (continued)Introduction (continued) Search of nearby conformational Search of nearby conformational
space and sequence spacespace and sequence space 2 methods of protein redesign 2 methods of protein redesign
(variation of backbone conformation (variation of backbone conformation and amino acid sequence)and amino acid sequence)
Development of procedure for Development of procedure for identifying low free energy sequence-identifying low free energy sequence-structure pairs that iterates between structure pairs that iterates between sequence optimization and structure sequence optimization and structure predictionprediction
Result: 93 residue protein with Result: 93 residue protein with topology not in PDBtopology not in PDB
Structure to SequenceStructure to Sequence
RosettaDesign predicts a amino acid RosettaDesign predicts a amino acid sequence from a desired structure.sequence from a desired structure.
Input and OutputInput and Output Input a structure you would like to Input a structure you would like to
createcreate Output a amino acid sequence that will Output a amino acid sequence that will
produce the structure with low free produce the structure with low free energyenergy
Sequence to StructureSequence to Structure
RosettaDesign is used to predict RosettaDesign is used to predict protein structure from protein protein structure from protein residue sequence. residue sequence.
Input and OutputInput and Output Input Amino Acid sequenceInput Amino Acid sequence Output a Predicted near minimum free Output a Predicted near minimum free
energy structureenergy structure
How does Top7 compare to How does Top7 compare to proteins in nature?proteins in nature?
Folding Folding Stability Stability StructureStructure
StabilityStability
Thermally stableThermally stable CD Spectrum at CD Spectrum at
9898˚C is nearly ˚C is nearly indistinguishable indistinguishable from that at 25˚Cfrom that at 25˚C
Folding of Top7Folding of Top7
At intermediate At intermediate concentrations (~5 M) concentrations (~5 M) of guanidine of guanidine hydrochloride (GuHCl) hydrochloride (GuHCl) Top7 unfolds Top7 unfolds cooperativelycooperatively
Steep transition in Steep transition in chemical denaturation chemical denaturation is characteristic of the is characteristic of the two-state unfolding two-state unfolding expected for small, expected for small, two-state, monomeric two-state, monomeric single-domain proteinsingle-domain protein
StructureStructure
Nuclear Overhauser Nuclear Overhauser effect spectroscopy effect spectroscopy (NOESY) and (NOESY) and heteronuclear single heteronuclear single quantum coherence quantum coherence (HSQC) exhibit (HSQC) exhibit features features characteristic of characteristic of protein with protein with substantial beta-sheet substantial beta-sheet content content
Comparing Top7 to modelComparing Top7 to model
CrystallizationCrystallization Top7 yielded crystals that diffracted to 2.5 Top7 yielded crystals that diffracted to 2.5 ÅÅ Strong molecular replacement (MR) solution Strong molecular replacement (MR) solution
to phase problemto phase problem This suggest design model very close to true This suggest design model very close to true
structurestructure
Top7 crystal like the model was also Top7 crystal like the model was also judged to be a novel topology by TOPS judged to be a novel topology by TOPS server server
Comparison of model (blue) Comparison of model (blue) and the solved x-ray structure and the solved x-ray structure
(red)(red)
ImplicationsImplications
Atomic Level Accuracy Atomic Level Accuracy (RMSD = 1.17 (RMSD = 1.17 ooA) in A) in de novo Protein de novo Protein DesignDesign
Validation of Accuracy Validation of Accuracy of Potential Functionsof Potential Functions
ReasonsReasons
Optimization of Sequence and StructureOptimization of Sequence and Structure
No Functional ConstraintsNo Functional Constraints
Extensive OptimizationExtensive Optimization
No kinetics No kinetics
Possible Future ImpactsPossible Future Impacts
Synthetic proteinsSynthetic proteins
Protein Therapeutics and Molecular Protein Therapeutics and Molecular MechanicsMechanics