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